Genotoxicity evaluation of individuals working with photocopying machines

Mutation Research 563 (2004) 151–158

Genotoxicity evaluation of individuals working with photocopying machines K. Iravathy Gouda,1 , Q. Hasana,b , N. Balakrishnac , K. Prabhakar Raod , Y.R. Ahujae,∗ b

a Department of Genetics, Mahavir Hospital and Research Center, Masab Tank, Hyderabad 500004, India Department of Genetics and Molecular Medicine, Kamineni Hospitals, L.B. Nagar, Hyderabad 500068, India c Department of Statistics, National Institute of Nutrition, Tarnaka, Hyderabad 500007, India d Department of Genetics, Osmania University, Hyderabad 500007, India e Department of Genetics and Molecular Medicine, Vasavi Medical Research Center, 6-1-91 Khairathabad, Hyderabad 500004, India

Received 26 August 2003; received in revised form 2 July 2004; accepted 2 July 2004

Abstract Photocopying machines are a common sight in the cities of India. There is ample evidence showing that the components of toners individually or in the form of a complex mixture are genotoxic. Toxic components of the photocopiers are from their emissions, toners and extremely low frequency electromagnetic fields (ELF-EMFs). In the present study micronucleus test (MNT) on buccal epithelial cells, cytokinesis block micronucleus (CBMN) assay and chromosomal aberration analysis on peripheral blood mononuclear cells was performed on 98 workers occupationally involved in photocopying and 90 age and sex matched controls. The results showed a significant increase in the frequency of MN in buccal epithelial cells and peripheral blood lymphocytes, as well as chromosomal aberrations in the exposed as compared to the control subjects. © 2004 Elsevier B.V. All rights reserved. Keywords: Photocopying machines; Photocopying toners; Micronucleus test; Chromosomal aberrations

1. Introduction ∗

Corresponding author. Tel.: +91 40 23323235; fax: +91 40 23315822. E-mail address: [email protected] (Y.R. Ahuja). 1 Present address: Department of Molecular Diagnostics, Indraprastha Apollo Hospitals, Saritha Vihar, New Delhi, India. 1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.07.001

Photocopying has become one of the common modes of employment in urban India. Photocopiers are being extensively used in offices, educational institutions and commercial establishments. Toner (ink) is used in the photocopiers to produce an image on pa-

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per or transparency. Two essential components of dry toners are colorants (most common being carbon black) and binder resins. In addition, the components of toners include polycyclic aromatic hydrocarbons (PAHs) and styrene [1]. The operators are exposed to the toners (while reloading the machines) and to toxic gases like ozone, nitrogen dioxide, volatile organic compounds (VOCs) and extremely low frequency electromagnetic fields (ELF-EMFs) during their operation [2–6]. Rosenkranz et al. [1] and Lofroth et al. [7] reported the mutagenic potential of photocopying toners in Salmonella tester strain TA98 in the absence of rat liver metabolic activation. The results showed that these toners could cause frame shift mutations. Mutagenic effects of components of photocopying toners and their emissions in animals and humans have also been reported [8–11]. We earlier conducted comet assay on individuals working with photocopying machines and the results showed that there was increased DNA damage and reduced repair efficiency in these subjects when compared to the controls [12]. In the present study we analyzed the genotoxic effects using micronucleus test (MNT) in buccal epithelial cells, cytokinesis block micronucleus assay (CBMN) and chromosomal aberration test (CA) in peripheral blood lymphocytes of workers operating photocopying machines for their livelihood.

2. Materials and methods 2.1. Subjects Ninety eight males working with photocopying machines for more than a year were included in this study. The exposed subjects were in the age group of 17–43 years and their duration of work varied from 1 to 10

years at the rate of 8–10 h per day for 6 days a week. Ninety males matched for age and socioeconomic status, working in different professions (like clerks, attenders and students) were randomly selected from the population to serve as controls. Information on general health status, reproductive history, diet, social habits and medication used during the last 3 months was collected in a well designed proforma from the exposed and control subjects (Table 1). 2.2. Photocopying machines The make of photocopying machines used by most of the individuals was Canon (Model Nos. 270, 3050, 4050, 6130, 6060, 8570), and were supplied with sachets of Recon Toner for Canon photocopying machines in powder form. Other machines were Sharp (Model No. 1101), Mita (Model Nos. 122, 213) and Modi (Model Nos. 1025, 1038, 5223), with matching brands of toners in powder form. 2.3. Sample collection Buccal epithelial cell and heparinized peripheral blood samples were obtained from all the individuals. 2.4. Micronucleus test (MNT) in buccal epithelial cells Micronucleus is a small extranucleus separated from the main one, generated during cell division by lagging chromosome(s) or by chromosome fragments. MN is considered to be an indicator of carcinogenic process, especially in the oral region. The subjects were asked to rinse their mouth with water. Oral mucosa was swabbed with a moistened wooden spatula from both the cheeks, smeared evenly on two pre-cleaned slides and air-dried. The slides were

Table 1 Demographic details of samples studied Parameters

Sample size (exposed subjects)

Sample size (control subjects)

Age range (years)

Duration of work (years)

Sample size of exposed smokers/ control smokers

Sample size of exposed nonsmokers/ control nonsmokers

Micronuclei in buccal epithelial cells Micronuclei in blood lymphocytes Chromosomal aberrations

98

90

17–43

1–10

51/79

47/11

98

90

17–43

1–10

51/79

47/11

55

37

17–43

1–10

28/33

27/4

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Table 2 Mean (%) ± S.E. of micronuclei (%MN) and micronucleated cells (%MN cells) in buccal epithelial cells and peripheral blood lymphocytes of exposed and control subjects Groups Buccal cells Exposed Control Lymphocytes Exposed Control ∗∗

Sample size

No. of cells

Total no. of MN

Mean% + S.E. of MN

Total no. of MN cells

Mean% + S.E. of MN cells

98 90

196000 180000

1230 690

0.63 ± 0.03∗∗ 0.38 ± 0.02

356 258

0.18 ± 0.01∗∗ 0.14 ± 0.01

98 90

196000 180000

902 463

0.46 ± 0.02∗∗ 0.26 ± 0.02

695 406

0.36 ± 0.02∗∗ 0.23 ± 0.02

P < 0.01.

fixed in Carnoy’s fixative (methanol and glacial acetic acid 3:1) and stained with 2% Giemsa solution for 10 min, rinsed in distilled water and air-dried. The cells were analyzed under a magnification of 400× using a light microscope. Two thousand buccal epithelial cells were screened per subject for the presence of micronuclei.

licate, flame dried and stained with 2% Giemsa solution for 10 min, rinsed in water and air-dried. The cells were analyzed under a magnification of 400× using light microscope. 2000 binucleated cells were scored per subject for micronuclei by the method of Fenech and Morley [13]. 2.6. Chromosome aberration (CA) test

2.5. Cytokinesis block micronucleus (CBMN) in peripheral blood lymphocytes MN is a chromatin containing structure in the cytoplasm surrounded by a membrane without any detectable link to the cell nucleus. Cytochalasin-B prevents the cells from completing cytokinesis resulting in the formation of binucleate cells, which enables the detection of MN. Whole blood cultures were set up in duplicate in RPMI1640 supplemented with fetal calf serum and antibiotics. The cultures were incubated for 72 h at 37 ◦ C. Cytochalasin-B solution (6 ␮g/ml) was added at the 44th hour after initiation of culture and incubated further for another 28 h at 37 ◦ C and then harvested. The cultures were treated with hypotonic solution (0.56% KCl) for 20 min at 37 ◦ C. After centrifugation the supernatant was discarded and the pellet fixed in 3:1 methanol:acetic acid. The slides were prepared in trip-

Chromosomal aberrations (CA) are the microscopically visible spectrum of DNA changes generated by different repair mechanism of DNA double strand breaks. CA breakpoints occur preferentially in active chromatin. Telomere-repeat like sequences play an important role in the formation of CA. Peripheral blood was initiated in RPMI 1640 media supplemented with fetal calf serum and antibiotics and incubated for 48 h at 37 ◦ C. After colchicine (0.004 mg/ml) treatment for 1 h and hypotonic (0.56%KCl) treatment for 30 min, the cells were fixed in 3:1 ratio of methanol and glacial acetic acid [14]. Slides prepared were flame dried and an attempt was made to analyze 100 metaphases per individual for chromosomal aberrations. Since the mitotic index of all the individuals was not very good only the first 70 well spread metaphase plates analyzed were included in the study.

Table 3 Mean (%) ± S.E. of cells with chromosomal aberrations and types of aberrations in peripheral blood lymphocytes of exposed and control subjects Groups

Sample size

No. of metaphases

Aneuploid cells

Cells with chromosomal aberrations

Exposed Control

55 37

3850 2590

6.16 ± 0.3∗∗ 1.89 ± 0.2

9.52 ± 0.62∗∗ 5.86 ± 0.36

∗∗

P < 0.01.

Types of aberrations Gaps

Breaks

Fragments

3.20 ± 0.27∗∗ 2.03 ± 0.2

4.82 ± 0.37∗∗ 3.22 ± 0.26

1.47 ± 0.22∗∗ 0

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Table 4 Effect of years of work in exposed subjects (sample size is given in parenthesis) Groups (years)

%MN in buccal epithelial cells

%MN in peripheral blood lymphocyte

% aberrant cells

(a) 1–5 (b) 6–10 a vs. b

0.41 ± 0.03 (53) 0.88 ± 0.04 (45)

0.33 ± 0.18 (53) 0.61 ± 0.02 (45)

10.62 ± 0.80(32) 16.76 ± 1.32(23)

∗∗

∗∗

∗∗

∗∗

P < 0.01.

2.7. Statistical analysis

3.3. CA test

The data for each parameter for the group was pooled and mean ± S.E. calculated for each group. Student’s t-test and analysis of variance (ANOVA) were performed to evaluate the differences. Regression analysis was done to study the effect of confounding factors. Pearson’s correlation analysis was carried to validate the sensitivity of each parameter.

Since the mitotic index of all individuals was not the same, we included only the first 70 metaphases analyzed from each individual. All samples where the analyzable metaphases were less than 70, were not included in the data, hence the number of individuals included were 55 exposed and 37 controls. The mean of aberrant cells (9.52) and mean percentage of aberrations (13.19) in the peripheral blood lymphocytes of the exposed subjects were significantly higher than those (5.86 and 8.41 respectively) in the control subjects (Table 3). The common types of chromosomal abnormalities seen were gaps, breaks, acentric fragments and aneuploid cells (Table 3).

3. Results 3.1. MNT There was a significant increase in the mean percentage of micronuclei in buccal epithelial cells of the exposed subjects (0.63) as compared to the control subjects (0.38) (Table 2).

3.4. Confounding factors Of the various confounding factors studied duration of work and smoking had significant effects.

3.2. CBMN assay

3.5. Duration of work (years of exposure)

The mean percentage of micronuclei in blood lymphocytes of the exposed subjects (0.46) was also significantly higher than in the control subjects (0.26) (Table 2).

The duration of work was divided into two groups: (a) 1–5 years and (b) 6–10 years. There was a significant increase in the micronucleus frequency in buccal epithelial cells as well as lympho-

Table 5 Effect of smoking in exposed and control subjects (sample size is given in parenthesis) Groups Exposed workers (a) Smokers Nonsmokers a vs. b Controls (a) Smokers Nonsmokers a vs. b ∗∗

P < 0.01.

%MN in buccal epithelial cells

%MN in peripheral blood lymphocyte

% aberrant cells

0.88 ± 0.04 (51) 0.42 ± 0.03 (47)

0.60 ± 0.02 (51) 0.30 ± 0.01 (47)

16.62 ± 0.19 (28) 9.62 ± 0.62 (27)

0.36 ± 0.01 (79) 0.06 ± 0.009 (11)

0.29 ± 0.01 (79) 0.05 ± 0.01 (11)

9.26 ± 0.34 (33) 1.42 ± 0 (4)

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

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Fig. 1. Inter-individual variation in micronucleus frequency in buccal epithelial cells was observed in both exposed and control subjects, however, the spread towards higher number of cells with micronuclei was seen in exposed when compared to controls.

cytes (CBMN assay) and in chromosomally aberrant cells with increase in the duration of work in the exposed subjects (Table 4). 3.6. Smoking The smokers of exposed and control subjects showed a significant increase in mean percentage of micronuclei in buccal epithelial cells, peripheral blood lymphocytes (CBMN assay) and chromosomally aberrant cells as compared to nonsmokers of both the groups (Table 5). Further, the comparison between smokers of exposed and control subjects showed that the exposed smokers had a significantly higher values for all the above-mentioned biomarkers as compared to the control smokers (Table 5).

3.7. Inter-individual variations Inter-individual variation was studied for micronucleus frequency in buccal epithelial cells and CBMN assay in peripheral blood lymphocytes in exposed and control subjects. It showed a greater spread in the exposed as compared to the control subjects (Fig. 1). 3.8. Correlation analysis Pearson’s coefficient of correlation was carried out between the values of different parameters to assess the extent of relationship between the micronucleus test in buccal epithelial cells, peripheral blood lymphocytes (CBMN assay) and chromosomal aberrations in the exposed and control subjects. It clearly showed a significantly positive correlation between all the tests

Table 6 Pearson’s correlation analysis in exposed and control subjects Groups Exposed Micronuclei in buccal epithelial cells Micronuclei in lymphocytes Aberrant cells Control Micronuclei in buccal epithelial cells Micronuclei in lymphocytes Aberrant cells ∗∗

P < 0.01.

Micronuclei in buccal epithelial cells

Micronuclei in lymphocytes

Aberrant cells

– 0.67∗∗ 0.59∗∗

0.67∗∗ – 0.67∗∗

0.59∗∗ 0.67∗∗ –

0.55∗∗ 0.63∗∗ 0.43∗∗

0.54∗∗ 0.67∗∗ 0.57∗∗

0.71∗∗ 0.64∗∗ 0.63∗∗

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employed to study the exposed and control subjects (Table 6).

4. Discussion Constant exposure of humans to toxic compounds in the workplace may lead to mutagenic/carcinogenic effects. In the present study the individuals working with photocopying machines could be exposed either by nasal or oral inhalation of the VOCs emanating during running of the machines, as well as, the EMFs generated, or by physical contact with toners during reloading the machines. The symptoms due to such exposures may result in short term and/or long-term effects. Short-term effects shown by the individuals included in the present study were headache, nausea, fatigue, breathing problem, eye irritation, cough, sneezing and general physical discomfort. Mullin et al. [9] reported similar symptoms in the workers occupationally exposed to high-speed print copying equipment. The symptoms observed in these workers could be due to the exposure to VOCs released from these machines in significant amounts during operation. To assess long-term effects genotoxicity studies were carried out. In the present study, MNT was carried out in buccal epithelial cells as well as the peripheral blood lymphocytes (CBMN). The reason for selecting buccal epithelial cells for studying micronucleus frequency was that the personnel working with photocopiers, during running and reloading of the machines, may frequently inhale nasally or orally the VOCs emanating from the machines. In occupational exposures epithelial cells are in direct route of airborne pollutants and they can metabolize proximate carcinogens [15,16]. In epidemiological biomarker studies, genetic changes in these epithelial cells and peripheral blood lymphocytes are of particular interest because these cells are frequently the targets of toxic agents. Increase in the frequency of cells with micronuclei and chromosomal aberrations seen in the present study reflects a sustained mutagenesis in the epithelial cells and peripheral blood lymphocytes due to the presence of mutagenic components of toners. During reloading and operation of the photocopying machines individuals are exposed to carbon black, styrene, PAHs, ozone, nitrogen dioxide and VOCs, as well as ELF-EMFs.

Their interaction with DNA has resulted in different types of chromosomal aberrations seen in the present study. Some of these components, either directly or through their metabolites, may have formed DNA adducts. These adducts during faulty repair process could probably open up and form single strand breaks which after replication could result in double strand breaks/micronuclei. Similar results were reported in workers occupationally exposed to formaldehyde [17]. In our exposed subjects chromatid-type of aberrations were more predominant than chromosome type of aberrations. Chemicals are known to cause chromatidtype damage in vitro, however, a number of in vivo studies also exhibited chromatid-type aberrations in spite of the fact that most chemical mutagens are Sphase dependent [18–20]. We feel that the increased chromatid damage observed by us is due to the persistence of adducts formed, until the S-phase of stimulated lymphocytes, to induce strand breaks. The other reason could be that single base pair/nucleotide changes may be induced by the genotoxic agents emanating from the photocopying machines and these appear as chromatid aberrations when there is failure of repair or errors in repair during in vitro proliferation. Of the various confounding factors studied, duration of work and smoking showed significant effect (Tables 4 and 5). Duration of exposure has also shown a positive effect in many other studies. For example, workers of phosphate fertilizer factory, out-door painters, and those exposed to sulphur dioxide and lowdose ionizing radiation showed increased frequency of MN and chromosomal aberrations with increase in duration of work suggesting a cumulative genotoxic effect [21–26]. Smoking exhibited a genotoxic effect in the exposed as well as control subjects. Further when comparison was made between the exposed smokers and the control smokers, the exposed smokers showed increased micronucleus frequency in the buccal epithelial cells and peripheral blood lymphocytes and chromosomal aberrations than the control smokers (Table 5) suggesting a synergistic effect of smoking and exposure to toxicants present in the toners and/or released during operation of the photocopying machines. In bidi and hooka smokers also a significant increase in cytogenetic parameters has been reported [26,27]. Inter-individual variation was studied for micronucleus frequency in buccal epithelial cells and CBMN

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assay in peripheral blood lymphocytes in exposed and control subjects. Greater number of cells with micronuclei were seen in exposed as compared to the control subjects, Fig. 1 shows inter-individual variability in buccal epithelial cells. Similar results were seen in the dose response of the number of micronuclei in cytokinesis-blocked lymphocytes after in vitro irradiation of whole blood with X-rays in the dose range 0–4 Gy in a heterogeneous population of 10 donors [28]. Pearson’s correlation analysis showed a positive correlation between the biomarkers used (Table 6). The present findings highlight the importance of using multiple DNA damage detection parameters in studying a genotoxic effect, since this information provides an increased degree of confidence for identification of the positive response. A significant correlation was also reported between micronucleus frequency, and SCEs in radiation induced human cervix carcinoma cell lines [29]. To the best of our knowledge this is the first detailed study, which has been carried out on a large sample size in subjects occupationally using photocopying machines, employing sensitive biomarkers in order to evaluate the genetic effects of combined exposure to toner chemicals, VOCs and ELF-EMFs.

Acknowledgements Financial support provided by Mahavir Hospital and Research Center to KIG is acknowledged. Advice of Dr. K. Visveswara Rao and Dr. Ravindra Tiwari is appreciated. We thank Mrs. Aruna and Mr. Tuljaram for secretarial assistance.

References [1] H.S. Rosenkranz, E.D. McCoy, D.R. Sanders, M. Butles, D.K. Kiriazides, R. Mermelstein, Nitropyrenes: isolation, identification, and reduction of mutagenic impurities in carbon black and toners, Science 209 (1980) 1039–1042. [2] M.D. Selway, R.J. Allen, R.A. Wadden, Ozone production from photocopying machines, Ann. Ind. Hyg. Assoc. 41 (1980) 455–459. [3] M.L. Lindobohm, M. Hietanen, Magnetic fields of video display terminals and pregnancy outcome, J. Occup. Environ. Med. 37 (1995) 952–956.

157

[4] S.K. Brown, Assessments of pollutant emissions from dry photocopiers, Indoor-Air 9 (1999) 259–267. [5] A.B. Stefaniak, P.N. Breysse, M.P. Murray, B.C. Rooney, J. Schaefer, An evaluation of employees to volatile organic compounds in three photocopy centers, Environ. Res. 83 (2000) 162–173. [6] Tuomi, B. Engstrom, R. Niemela, J. Svinhufvud, K. Reijula, Emission of ozone and organic volatiles from a selection of laser printers and photocopiers, Appl. Occup. Environ. Hyg. 15 (2000) 629–634. [7] G. Lofroth, E. Hefner, I. Alfhelm, M. Moller, Mutagenic activity in photocopiers, Science 209 (1980) 1037–1039. [8] P. Venier, G. Tecchio, E. Clonfero, A.G. Levis, Mutagenic activity of carbon black dyes used in the leather industry, Mutagenesis 2 (1987) 19–22. [9] L.S. Mullin, A.W. Ader, W.C. Daughtrey, D.Z. Frost, M.R. Greenwood, Toxicology update isoparaffinic hydrocarbons: a summary of physical properties, toxicity studies and human exposure data, J. Appl. Toxicol. 10 (1990) 135–142. [10] H. Hohensee, U. Flowerday, J. Oberdick, Zum Emissionsverhalten von Farbfotokopierger¨aten und Farblaserdruckern, Die BG Nr. 11S (2000) 659–662. [11] E. Nies, H. Blome, H. Br¨uggemann-Prieshoff, Charakterisierung von Farbtonern und Emissionen aus Farbfotokopierern/Farblaserdruckern, Gefahrstoffe-Reinhalt 60 (2000) 435– 441. [12] K.I. Goud, K. Shankar, B. Vijayashree, Y.R. Ahuja, Genotoxic effects in individuals working with photocopying machines, Int. J. Hum. Genet. 1 (2001) 139–143. [13] M. Fenech, A. Morley, Measurement of micronuclei in lymphocytes, Mut. Res. 147 (1985) 29–36. [14] P.S. Moorhead, P.C. Nowell, W.J. Mellman, D.M. Battips, D.A. Hungerford, Chromosome preparation of leukocytes cultured from human peripheral blood, Exp. Cell Res. 20 (1960) 613–616. [15] L. Zhang, D. Mock, Gamma-glutamyl transpeptidase activity in superficial exfoliated cells during hamster buccal pouch carcinogenesis, Carcinogenesis 10 (1989) 857– 860. [16] J.G. Stone, N.J. Jones, A.D. McGregor, R. Waters, Development of human biomonitoring assay using buccal mucosa: comparison of smoking related DNA adducts in mucosa versus biopsies, Cancer Res. 55 (1995) 1267–1270. [17] G. Motykiewicz, J. Michalska, J. Pendzich, E. Malusecka, M. Strozyk, E. Kalinowska, D. Butkiewicz, D. Mielzynska, A. Midro, R.M. Santella, A molecular epidemiology study in women from Upper Silesia Poland, Toxicol. Lett. 96 (1998) 195–202. [18] Jelmert, I.L. Hansteen, S. Langard, Chromosome damage in lymphocytes of stainless steel welders related to past and current exposure to manual metal arc welding fumes, Mut. Res. 320 (1994) 223–233. [19] D. Anderson, A. Yardley-Jones, C. Vives-Bauza, W. Chuaanusorn, C. Cole, J. Webb, Effect of iron salts, haemosiderins, and chelating agents on the lymphocytes of a thalassaemia patient without chelation therapy as measured in the comet assay, Teratogen. Carcin. Mut. 20 (2000) 251–264.

158

K.I. Goud et al. / Mutation Research 563 (2004) 151–158

[20] T. Caldes, P. Perez-Segura, A. Tosar, M. de la Hoya, E. DiazRubio, Microsatellite instability correlates with negative expression of estrogen and progesterone receptors in sporadic breast cancer, Teratogen. Carcin. Mut. 20 (2000) 283–291. [21] J.S. Yadav, N. Seth, Effect of polycyclic aromatic hydrocarbons on somatic chromosomes of coal tar workers, Cytobios 93 (1998) 165–173. [22] J.S. Yadav, Kaushik, Genotoxic effects of ammonia exposure on workers in a fertilizer factory, Indian J. Exp. Biol. 35 (1996) 487–492. [23] Z. Meng, B. Zhang, Chromosomal aberrations and micronuclei in lymphocytes of workers at a phosphate fertilizer factory, Mut. Res. 393 (1997) 283–288. [24] D. Pinto, J.M. Ceballos, G. Garica, P. Guzman, L.M. Del Razo, E. Vera, H. Gomez, A. Garcia, M.E. Gonsebatt, Increased cytogenetic damage in outdoor painters, Mut. Res. 467 (2000) 105–111.

[25] C.K. Chitra, H. Vishwanathan, E. Deepa, M.V. Rani, Cytogenetic monitoring of men occupationally exposed to airborne pollutants, Environ. Pollut. 112 (2001) 391– 393. [26] J.S. Yadav, S. Thakur, Cytogenetic damage in bidi smokers, Nicotine Tobacco Res. 2 (2000) 97–103. [27] J.S. Yadav, S. Thakur, Genetic risk assessment in hookah smokers, Cytobios 10 (2000) 101–113. [28] H. Thierens, A. Vral, L. de Ridder, Biological dosimetry using the micronucleus assay for lymphocytes: interindividual differences in dose response, Health Phys. 61 (1991) 623– 630. [29] A.M. Eastham, J. Atkinson, C.M. West, R. Perrin-Nadif, M. Dusch, C. Koch, Relationships between clonogenic cell survival, DNA damage and chromosomal radiosensitivity in nine human cervix carcinoma cell lines, Int. J. Radiat. Biol. 77 (2001) 295–302.