genotoxicity study in lymphocytes of offset printing workers

10 H. AKSOY ET AL.

Copyright © 2005 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2006; 26: 10–15

JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 2006; 26: 10–15Published online 12 September 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jat.1098

Genotoxicity study in lymphocytes of offset printingworkers

Hüseyin Aksoy,1 Serkan Yılmaz,1 Mustafa Çelik,2 Deniz Yüzba6ıoglu1* and Fatma Ünal1

1 Gazi Üniversitesi, Fen-Edebiyat Fakültesi, Biyoloji Bölümü, 06500 Teknikokullar, Ankara, Turkey2 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 ex-

changes (SCEs), chromosome aberrations (CAs) and micronuclei (MN) as biomarkers in peripheral lymphocytes of 26 vol-

unteers (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)

* Correspondence to: Deniz Yuzbasıoglu, Gazi Üniversitesi, Fen-EdebiyatFakultesi, Biyologi Bölümü, 06500 Teknikokullar, Ankara, Turkey.E-mail: [email protected]

Introduction

In recent years, there has been an increased problemof human exposure to potentially toxic chemicals inthe work place. A large volume of epidemiological datadeals with potential cancer risks in printing processes.Svensson et al. (1990) reported that a significant increasein cancers of the respiratory tract was observed amongrotogravure printers. Aerosols from high speed rotaryprinting machines consist of ink mists, which are believedto induce lung cancer (Leon et al., 1994). In additionsome studies reported that kidney cancer, urinary bladdercancer and leukemia were shown among printing workers(IARC, 1996).

Offset printing workers are exposed to offset printingdyes and thinner. Offset printing dyes contain cobalt andhydroquinone. Cobalt is a heavy metallic chemical ele-ment. It is a member of group VIII of the periodic table.It is used for cancer therapy and in industry for detectingflaws in metal parts. The overall genetic evaluation wasthat cobalt and its compounds are possibly carcinogenicto humans (group 2B) (IARC, 1991). In the same evalu-ation, cobalt (II) compounds were reported to induceDNA damage, DNA protein cross links, gene mutations,sister chromatid exchanges and aneuploidy in in vitro

studies on animal and human cells. There was someevidence that cobalt (II) compounds could also induceaneuploidy in vivo in Syrian hamster bone marrow andtestes (IARC, 1991). Another chemical used in printing,

hydroquinone, is extensively used as a reducing agentand as a photographic developer. It is a skin-lighteningagent and is used in cosmetics, hair dyes and medicalpreparations (WHO, 1996). Knadle (1985) reportedthat hydroquinone induced sister chromatid exchanges(SCEs) at relatively high concentrations in humanlymphocytes. Stillman et al. (1999) reported that hydro-quinone induced hypoploidy in a human lymphoblastoidcell line GM09948. Offset printing workers also usewidely an industrial thinner containing toluene (about65%) for the cleaning of the offset machines. Bauchingeret al. (1982) and Schmid et al. (1985) reported an in-creased incidence of SCEs in individuals occupationallyexposed to toluene. A high incidence of chromosomalaberrations (CA) and sister chromatid exchanges in thelymphocytes of rotogravure printing workers has beenreported. The CA and SCE frequencies were higher inworkers than in the control groups (Pelclova et al., 1990;Hammer et al., 1998). However, some studies in the lit-erature showed that there was no relationship betweenchronic occupational exposure to toluene and increasedincidence of chromosomal aberration (Haglung et al.,1980; Maki-Paakkanen et al., 1980; Richer et al., 1993).

Genotoxicity biomarkers have received considerableinterest as tools for detecting human genotoxic expo-sure and effects. The largest databases are availablefor CA; a high CA level has been associated with anincreased cancer risk while the frequencies of SCEs andmicronucleus (MN) were not associated with cancer risk(Hagmar et al., 1998a,b; Bonassi et al., 2000). The induc-tion of SCEs has been described as a rapid and sensitiveend-point for testing mutagenicity. An increase ofSCE frequency can be an indicator of persistent DNA

GENOTOXICITY STUDY IN OFFSET PRINTING WORKERS 11


damage (Palitti et al., 1982; Cardoso et al., 2001). Thecytokinesis-blocked micronucleus assay is used to detectacentric chromosome fragments or whole chromosomesleft upon nucleus division and visible as small additionalnuclei in the cytoplasm. Hence, the appearance of micro-nuclei should point to clastogenic and/or aneugeniceffects (Giri et al., 2002).

This paper analysed cytogenetic damage in peripheralblood lymphocytes of offset printing workers by usingCAs, SCEs and micronucleus assay (MN). Changes incell proliferation kinetics through the replication index(RI) and cytotoxic effect by means of the mitotic index(MI) were also evaluated.

Material and Methods

Twenty six volunteers (14 exposed, 12 controls) werestudied in this work. The volunteers were selectedaccording to their ages and smoking habit. Three agegroups were selected: 16–25, 26–35 and 36–45. Noneof the volunteers had any problems relating to health,alcohol and drug consumption. Six of the exposedworkers were smokers, the others were nonsmokers.Their ages were in the range 18–45 years and averaged27.64 years. The mean duration of employment was10.36 years (min. 1 year, max. 30 years). The workingperiod was 45 h per week. A total of 12 male blooddonors from the Gazi University of Ankara were used asa control group. This group consisted of six smokers andsix nonsmokers, aged 22–38 years (average 28.70 years).The preparations of cells were made by the followingprocedures.

SCE and CA Analysis

Human peripheral blood cells were used as the test sys-tems. Heparinized blood (1/10: 2 ml venous blood con-taining 0.2 ml heparin) was collected and stored at +4 °Cfor 30 min. The whole blood was added to 2.5 mlchromosome medium B (Biochrom) supplemented with10 µg ml−1 bromodeoxyuridine. The cultures were incu-bated at 37 °C for 72 h. 0.06 µg ml−1 colchicine wasadded 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 hypotonicsolution of 0.075 M KCl for 30 min at 37 °C. Followingthis process, the cells were centrifuged again and fixedin a cold methanol:acetic acid (3:1) mixture for 35 minat +4 °C. At the end of this procedure, the cells weretreated with fixative two times. Then slides were made bydropping and air drying.

For chromosome aberrations, slides were stained with5% Giemsa (pH = 6.8) prepared in Sorensen buffer solu-tion, for 20–25 min, and then washed in distilled water,

dried at room temperature and mounted with depex. Forthe SCE study, the slides were stained with Giemsaaccording to the method of Speit and Houpter (1985)with some modifications.

The mitotic index (MI) was determined by scoring1000 cells from each donor. Chromosomal abnormalitieswere scored from 100 well-spread metaphases per donor.For the occurrence of the number of SCEs, a total of25 cells from each donor under second metaphases wasscored. In addition, 100 cells from each donor werescored for the determination of the replication index (RI).The RI, calculated according to the following formula

[1 × M1] + [2 × M2] + [3 × M3]/N

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 mlchromosome medium B (Biochrom). The cultures wereincubated at 37 °C for 72 h. At 44 h after beginning theculture, cytocalasin-B (5.2 µg ml−1) was added to eachculture. Then, the cells were harvested by centrifugation(1000 rpm, 10 min), and the pellets were resuspended ina hypotonic solution of 0.075 M KCl for 5 min at +4 °C.The cells were again centrifuged and fixed in the coldmethanol:acetic acid (3:1) mixture for 15 min. Thefixation procedure was applied three times. Into the lastfixative, 1% formaldehyde was added to preserve thecytoplasm. The slides were made by dropping and airdrying. For the MN analysis, the slides were stained with5% Giemsa (pH = 6.8), prepared in Sorensen buffersolution, for 20–25 min, washed in distilled water,dried at room temperature and mounted with depex.Micronuclei were scored from 1000 binucleated cellsper donor.

Statistical Analysis

The significance between the percentage of abnormalcells, CA/cell, RI, MI and MN in cultures and their con-trols were determined using the z-test. The significancebetween mean SCE in treated cultures and their controlswere determined using the t-test.

Results

In this study, 26 volunteers were studied. Of these, 14were offset printing workers and 12 were controls. Asshown in the Fig. 1 and Table 1, the abnormal cell ratio

12 H. AKSOY ET AL.


was significantly increased compared with their controls.In the smoker workers the increase of abnormal cells washigher than in the nonsmokers. The CA/cell ratio signific-antly increased in exposed groups when compared withthe controls. Also the increase of CA/cell was higherin the smoker groups than in the nonsmoker groupsin the 26–35 and 36–45 age groups (Table 1). Theeight types of abnormalities recorded were chromatid andchromosome breaks, sister chromatid union, dicentricchromosomes, endoreduplication, fragment, polyploidyand chromatid exchange. Chromatid breaks and sisterchromatid union were observed as the most commonaberrations (Fig. 2).

The replication index (RI) was not affected in theworker groups but the mitotic index was decreasedsignificantly in all the age groups of nonsmokers(Table 2). The SCE frequency was significantly increasedin exposed groups when compared with their controls(Table 2, Fig. 3). In all exposed groups, the SCEfrequency was higher in the smoker workers than in thenonsmoker workers.

The micronuclei frequency was significantly increasedin all exposed groups except in the smoker workers

Table 1. Types and distribution of chromosome aberrations in offset printing workers according to smoking habitand age

Group Age Smoking No. of No. of cells Aberrations Abnormal CA/cell ± SEhabit subjects scored

B′ B″ SU DC ER F P CEcell ± SE (%)

Controls 16–25 Smoker 2 200 6 — — — — — — — 3.00 ± 1.21 0.030 ± 0.01Workers Smoker 3 300 23 6 16 9 1 1 — — 16.00 ± 2.12a 0.190 ± 0.02a

Controls 16–25 Nonsmoker 2 200 — — 3 — — — — — 1.50 ± 0.86 0.015 ± 0.01Workers Nonsmoker 3 300 29 3 12 15 2 1 — 1 18.33 ± 2.24a 0.210 ± 0.02a

Controls 26–35 Smoker 2 200 8 — — — — — — 2 2.50 ± 1.10 0.050 ± 0.01Workers Smoker 1 100 10 1 2 1 1 — — — 13.33 ± 3.40a 0.150 ± 0.04a

Controls 26–35 Nonsmoker 2 200 1 — 3 — — — — — 2.00 ± 0.98 0.020 ± 0.01Workers Nonsmoker 3 300 27 3 3 4 — 4 1 — 14.00 ± 2.00a 0.140 ± 0.06a

Controls 36–45 Smoker 2 200 6 — — 2 — — — — 2.00 ± 0.99 0.040 ± 0.01Workers Smoker 2 200 13 6 17 9 — 4 4 — 26.00 ± 3.10a 0.270 ± 0.03a

Controls 36–45 Nonsmoker 2 200 2 — 1 1 — 1 — — 2.50 ± 1.00 0.025 ± 0.01Workers Nonsmoker 2 200 15 3 2 2 — 2 — — 11.00 ± 2.21a 0.120 ± 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 peripheralblood lymphocytes of smoker and nonsmoker offsetprinting workers and their controls

Figure 2. Most common aberrations observed inoffset printing workers, (a) chromatid break (b) sisterchromatid union

of the 16–25 age group (Table 3). The frequency ofmicronuclei was significantly higher in exposed groupscompared with their controls in total (Fig. 4). In all



Table 2. Distribution of SCE, RI and MI in peripheral blood lymphocytes of offset printing workers according tosmoking habit and age

Groups Ages Smoking No. of No. of cells Min-max SCE/cell M1 M2 M3 RI ± SE MI ± SE (%)habit subjects scored (SCE) SCE ± SE

Controls 16–25 Smoker 2 50 2–12 6.28 ± 0.41 38 50 112 2.37 ± 0.05 3.20 ± 0.39Workers Smoker 3 75 5–20 9.97 ± 0.35b 48 108 144 2.32 ± 0.04 3.90 ± 0.35Controls 16–25 Nonsmoker 2 50 2–9 4.68 ± 0.23 40 57 103 2.32 ± 0.05 5.50 ± 0.51Workers Nonsmoker 3 75 5–15 8.75 ± 0.29b 46 93 161 2.38 ± 0.04 3.83 ± 0.35c

Controls 26–35 Smoker 2 50 1–14 5.68 ± 0.45 10 50 140 2.75 ± 0.04 4.20 ± 0.44Workers Smoker 1 25 5–20 9.48 ± 0.74b 16 25 59 2.43 ± 0.08 4.60 ± 0.66Controls 26–35 Nonsmoker 2 50 1–19 4.70 ± 0.46 42 81 77 2.18 ± 0.05 7.60 ± 0.59Workers Nonsmoker 3 75 4–17 8.51 ± 0.29b 54 105 141 2.29 ± 0.04 3.57 ± 0.34d

Controls 36–45 Smoker 2 50 5–23 8.20 ± 0.61 30 52 118 2.44 ± 0.05 4.50 ± 0.46Workers Smoker 2 50 5–20 9.80 ± 0.47a 69 66 65 1.98 ± 0.06 3.85 ± 0.43Controls 36–45 Nonsmoker 2 50 2–8 5.12 ± 0.29 44 70 86 2.21 ± 0.06 6.25 ± 0.54Workers Nonsmoker 2 50 6–15 9.20 ± 0.32b 29 62 109 2.40 ± 0.06 4.05 ± 0.44c

a Significant from the control P < 0.05 (t-test)b Significant from the control P < 0.01 (t-test)c Significant from the control P < 0.01 (z-test)d Significant from the control P < 0.001 (z-test)

Figure 3. Sister chromatid exchanges in peripheralblood of offset printing workers according to smokinghabit and their controls

Table 3. Distribution of MN in peripheral blood lymphocytes of offset printing workers according to smokinghabit and age

Groups Ages Smoking No. of BN cells Distribution of BN cells MN/cell (%)habit subjects scored according to the no. of MN

(1) (2) (3) (4)

Controls 16–25 Smoker 2 2000 8 0 0 0 0.40 ± 0.138Workers Smoker 3 3000 16 2 0 0 0.67 ± 0.148Controls 16–25 Nonsmoker 2 2000 4 0 0 0 0.20 ± 0.094Workers Nonsmoker 3 3000 15 1 1 0 0.67 ± 0.148a

Controls 26–35 Smoker 2 2000 10 0 0 0 0.50 ± 0.154Workers Smoker 1 1000 11 1 0 0 1.30 ± 0.357b

Controls 26–35 Nonsmoker 2 2000 2 0 0 0 0.10 ± 0.063Workers Nonsmoker 3 3000 20 4 0 0 0.93 ± 0.173b

Controls 36–45 Smoker 2 2000 4 0 0 0 0.20 ± 0.094Workers Smoker 2 2000 19 2 0 0 1.15 ± 0.236b

Controls 36–45 Nonsmoker 2 2000 8 2 0 0 0.60 ± 0.170Workers Nonsmoker 2 2000 18 1 0 0 1.00 ± 0.221a

a Significant from the control P < 0.05 (z-test)b Significant from the control P < 0.001 (z-test)

exposed groups (except the 16–25 age group), the MNfrequency was higher in the smoker workers than in thenonsmoker workers but this result was not statisticallysignificant.

Figure 4. MN in peripheral blood of offset printingworkers according to smoking habit and their controls

Discussion

In this study the CA, SCE and MN (except smokersof the 16–25 age group) frequency of exposed workers

14 H. AKSOY ET AL.


significantly increased in all the age groups comparedwith their controls. The MI value was significantlydecreased only in the nonsmoker groups. However, theRI value was not affected in workers when comparedwith the controls.

In this investigation, eight types of chromosomalaberrations were observed and it was found that themost common aberrations were chromatid breaks andsister chromatid union. Pelclova et al. (1990) reportedthat chromatid and chromosome breaks, gaps, chromatidand chromosome exchanges were observed in rotogravureprinting workers. Genotoxicity of toluene was inves-tigated in rotogravure printing plant workers and asignificant increase was observed only in chromatidbreaks (Pelclova et al., 2000).

Printing dyes used by offset printing workers containcobalt and hydroquinone. Some researchers have shownthat cobalt was possibly carcinogenic to humans (IARC,1991) and that hydroquinone was positive for cytogeneticeffects, which included induction of micronuclei (Yageret 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 thathydroquinone was cytotoxic, but did not induce chromo-somal aberrations in human lymphocytes culture in vitro.

Offset printing workers also use thinner for machinecleaning processes. Thinner contains a high amount oftoluene and some researchers have reported that occupa-tional exposure to toluene increased the incidence ofSCEs (Bauchinger et al., 1982; Schmid et al., 1985;Pelclova et al., 2000). In contrast, some studies showedthat there was no relationship between chronic occupa-tional exposure to toluene and increased incidence ofchromosomal aberration and sister chromatid exchanges(Haglung et al., 1980; Maki-Paakkanen et al., 1980;Richer et al., 1993). Pelclova et al. (1990) reported thatCA frequency was increased in rotogravure printingworkers compared with controls. In another study Ham-mer et al. (1998) reported that the SCE frequency wassignificant at a high confidence level in rotogravure print-ing workers. The authors pointed out a strong relationshipbetween individual toluene burden and the genotoxic riskof the exposed workers.

Many studies have been carried out to evaluate thecytogenetic effects of occupational exposure to varioushealth hazard chemicals in different field workers. Sardaset al. (1994) reported that the frequency of SCE washigher 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 showedthat the frequency of CA was higher, while the frequencyof the SCE was not in all the smoker-nonsmoker workers,than in the smoker-nonsmoker control groups. In addi-tion, the authors observed that there was no significantdecrease in the RI, but the MI was significantly lowerin exposed groups than in the controls. Bogadi-Sare

et al. (1997) reported that exposure to benzene andtoluene increased the CA frequency in the lymphocytesof workers. The genotoxicity study of Swedish paintindustry workers showed that there was no significantincrease in the frequency of CA and SCE (Haglung et al.,1980). Pastor et al. (2001) reported that a compar-ison between workers and controls did not reveal anystatistical significant difference in the MN frequency foreither lymphocytes or buccal cells in Greek farmers.Genotoxicity research in wooden furniture workers hasshown that micronuclei and SCE frequency in exposedgroups was higher than in the controls (Elavarasi et al.,2002). Iravathy Goud et al. (2004) reported that there isa significant increase in the frequency of MN in buccalepithelial cells and peripheral blood lymphocytes as wellas chromosomal aberrations in individuals working withphotocopying machines. A significant increase in the for-mation of SCE, CA and MN frequencies were reported inPb-Zn miners exposed to heavy metals (Bilban, 1998).

In the literature, several studies demonstrated that CA,SCE and MN frequencies generally increased in workersoccupationally exposed to the environmental contam-inants discussed above. Some of these investigationsrevealed that there is a relationship between age, smok-ing and increase in abnormalities (Maki-Paakkanen et al.,1980; Topaktas et al., 2002). Whereas, some investigatorsreported 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, SCEand MN frequency of the exposed workers were higherthan the controls. The CA frequency and CA/cell wasalmost equal in smoker and nonsmoker workers in the16–25 and 26–35 age groups but higher only in smokersof the 36–45 age group than in the nonsmokers. A strongrelationship between CA and smoking habit, age andexposure to pollutants was observed. In all exposedgroups, 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 wasnot associated with age. As a result, the present studyshowed that the CA analysis was more sensitive to envir-onmental contaminants than other cytogenetic end-points.It can be concluded from this study that chronic occupa-tional exposure to printing dyes and toluene leads to anincreased risk of genetic damage among offset printingworkers. The genotoxic and potential carcinogenic risksof printing dyes and toluene should be taken into accountin the process of offset printing.

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genotoxicity study in lymphocytes of offset printing workers

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