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  Table of Contents 
Year : 2012  |  Volume : 16  |  Issue : 1  |  Page : 34-37

Increase in DNA damage in lymphocytes and micronucleus frequency in buccal cells in silica-exposed workers

Department of Genetics, Ramakrishna Mission Seva Pratishthan, Vivekananda Institute of Medical Sciences, 99 Sarat Bose Road, Kolkata, India

Date of Web Publication13-Aug-2012

Correspondence Address:
Ajanta Halder
Department of Genetics, Vivekananda Institute of Medical Sciences, Ramakrishna Mission Seva Pratisthan, 99, Sarat Bose Road, Calcutta - 700 026
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0019-5278.99691

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The alkaline single cell gel electrophoresis (comet assay) was applied to study the genotoxic properties of silica in human peripheral blood lymphocytes (PBL). The study was designed to evaluate the DNA damage of lymphocytes and the end points like micronuclei from buccal smears in a group of 45 workers, occupationally exposed to silica, from small mines and stone quarries. The results were compared to 20 sex and age matched normal individuals. There was a statistically significant difference in the damage levels between the exposed group and the control groups. The types of damages (type I -type 1V) were used to measure the DNA damage. The numbers of micronuclei were higher in the silica-exposed population. The present study suggests that the silica exposure can induce lymphocyte DNA damage and produces significant variation of micronuclei in buccal smear.

Keywords: Comet assay, micronuclei, lymphocyte DNA damage, silica-exposed female workers

How to cite this article:
Halder A, De M. Increase in DNA damage in lymphocytes and micronucleus frequency in buccal cells in silica-exposed workers. Indian J Occup Environ Med 2012;16:34-7

How to cite this URL:
Halder A, De M. Increase in DNA damage in lymphocytes and micronucleus frequency in buccal cells in silica-exposed workers. Indian J Occup Environ Med [serial online] 2012 [cited 2022 Jul 5];16:34-7. Available from:

  Introduction Top

Crystalline silica has been classified as a human carcinogen by International Agency for Research on Cancer. [1] The distribution of silica in nature is similar to the distribution of carbon in organic matters. Silicon being very reactive does not remain in the element form, but combines either with oxygen and forms silica (SiO 2 ) or with oxygen and other elements and form silicates. Silica and silicates constitute the bulk of most kinds of rocks, clays, and sands. Exposure to large amount of free silica can pass unnoticed because silica is odorless, nonirritant, and does not cause any immediate noticeable effect and hence is confused with ordinary dust. Since the earth's crust contains about 12% free silica mostly in the form of quartz mining and tunneling are the occupations most closely related to the hazard of silica exposure. [2],[3]

To study the effects of occupational silica exposure on DNA, blood samples and buccal smears obtained from 45 stone mine and quarry workers in Siuri, West Bengal, were subjected to the alkaline single cell gel electrophoresis assay (comet assay) and micronuclear assay, respectively.

  Materials and Methods Top

Clearance for the study was obtained from the ethics committee of the Vivekananda Institute of Medical Sciences.

The subjects for the study were divided into two groups. (I) Mine and quarry workers with a history of 10-15 years of occupational silica exposure (n = 45) and (II) age-matched healthy control subjects (n = 20) with no known exposure to cytotoxic or genotoxic agents. The chosen 45 subjects were workers in small mines and quarries; their type of work was stone crushing, grinding, carrying chips, and extracting brick earths. All the subjects were from same area. Several mines are situated in a cluster within 2 km of one another. Informed consent was obtained after the nature of the procedure was fully explained.

5 ml heparinized (50 units/mol. sodium heparin) whole blood sample were collected for comet assay by venepuncture from each individual at the end of the work week. The samples were transported at 4°C to our laboratory on the same day and stored overnight at 4°C. There is no loss of cell viability at 4°C or room temperature up to 8 days according to Anderson et al. [4] The assays were completed within 3 days of sample collection. 20 μl whole blood was mixed with 1 ml ice cold RPMI 1640 in a microcentrifuge tube and lymphocytes were then isolated by Ficoll-Hypaque density gradient procedure. [5] The rest of the blood was used for routine analysis such as hemoglobin (Hb) concentration, packed cell volume (PCV), etc. The comet assay was performed by the method of Singh et al. [5] 110 μl 0.6% normal melting agarose (NMA) was coated on a fully frosted slide for a sound attachment and solidified for 5 min. The gel was covered by a 20 × 20 cover glass. 0.6% low melting point agarose mixed with 10 μl lymphocyte was coated over the first layer. A third, agarose, layer (75 μl) was coated over the second lymphocyte agarose layer at 4°C for another 10 min. After removal of the cover glass, the slides were immersed in the lysing solution (2.5 mol. NaCl, 100mM Na2EDTA, with freshly added 1% Triton-X100, and 10% DMSO) at 4C for 1 h. Slides were then placed on a horizontal electrophoresis stand filled with freshly prepared alkaline buffer (300 mM NaOH, 1 mM Na2EDTA, pH 13.0, 4°C) to a level of approximately 0.25 cm above the slide. The slides were allowed to set in this high pH buffer for 20-25 min to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was then conducted for 20 min at 25 V using an electrophoresis compact power supply (Bio Rad, USA) and the current was adjusted to 300 mA by raising or lowering the buffer. After electrophoresis the slides were washed gently to remove alkali and detergents by flooding them slowly with 0.4 M Tris buffer at pH 7.5. After 5 min the slides were stained by placing 20 mg/ml ethidium bromide in distilled water solution. Then the slides were covered with cover glasses.

Observation was made using 40× objective on a fluorescent microscope, (Nikon Microscope - Eclipse, E600 with Y-FL EPI-Fluorescence attachment, Japan) equipped with an excitation filter of 515-560 nm and a barrier filter 590 nm. One hundred cells were analyzed from each sample and the DNA damage was scored visually as described by Palus et al. [6] The cells were graded into five categories: no damage (Type 0), low-level damage [Figure 1], medium-level damage [Figure 2], high-level damage [Figure 3], and complete damage [Figure 4]. Analysis was performed by one slide reader, thus minimizing variability due to subjective scoring. For micronuclei study, subjects were asked to rinse their mouths with water and a premoistened wooden spatula was used to collect cells from the buccal mucosa. The spatula was applied to a clean microscopic slide. Smears were air dried and fixed in 80% methanol. Slides were stained in Giemsa stain [7] and 1000 cells were scored by the same person. Observed data were statistically evaluated by Student's "t" test.
Figure 1: Photograph of DNA low-level damage

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Figure 2: Photograph of DNA medium-level damage

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Figure 3: Photograph of DNA high-level damage

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Figure 4: Photograph of DNA complete damage

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  Results and Discussion Top

The silica-exposed workers were all females and nonsmokers. The main route of exposure was inhalation. The small mines are situated far from nearest town and there are no other factories or industrial entities nearby. Most of the workers had some clinical problems such as cough, common cold, fever, headache, and diarrhea [Table 1]. The Hb percentage was lower in those exposed than in the control group. The percentage of DNA damage was calculated by the percentage of each type (I, II, III, and IV) of comet. In case of the exposed population, different grades of DNA damage (Types II, III, and IV) were present; these were absent in the control group [Figure 5]. An increased percentage of micronuclei were observed in the exposed population [Table 2]. In this study, we evaluate the level of DNA damage in lymphocytes of a silica-exposed population using the comet assay. We also try to measure the genotoxic effect of such exposure. Alkaline single cell gel electrophoresis (comet assay) is a rapid, simple, and sensitive method for measuring and analyzing DNA single-strand breaks and alkali-labile sites. [5],[6],[8],[9],[10] This technique has been adopted as a useful tool in short-term genotoxicity and human biomonitoring studies due to its sensitivity in detecting genetic damage at the individual cell level and its potential application to virtually all eukaryotic cell types. [9],[10] In vitro toxicity testing by the comet assay has been demonstrated to be quantitatively reproducible. [11] Much work has been done on the association of silicosis with lung cancer [12],[13] and it was found that ultrafine silicon dioxide is cytotoxic and genotoxic in cultured human cells, while Yang et al. [14] studied the role of particle size, shape, and composition in genotoxicity and cytotoxicity of various nanomaterials including silica. Type 0 cells, without DNA damage, were predominant in the control group, while Types II, III, and IV cells were found in the exposed population. Increased DNA damage has also been seen in attendants exposed to low levels of benzene and other petroleum products [15] and in workers in a plastic factory, [16] whereas no statistically significant differences were observed in a study of workers exposed to 1, 3-butadiene. [17] The increase in micronuclei (MN) frequency may be related to occupational exposure to silica. Bolognesi et al. [18] found higher frequency of MN in traffic police officers exposed to lead, while Villarini et al. [2] found an increase in MN frequency amongst road tunnel workers. The MN assay in exfoliated cells is a useful technique for study of genotoxic effects of different carcinogens and mutagens in human populations. Karahalil et al. [19] reported that age, sex, and smoking habit did not influence the MN frequency. Stich and Rosin [20] showed the synergistic effect of smoking and alcohol consumption with the MN test on buccal cells and they found elevated frequencies of micronucleated buccal mucosal cells. A significant increase in MN frequencies was observed in submucous fibrosis patients by Stich and coworkers [21],[22] observed a nonsignificant increase in MN in exfoliated buccal and nasal cavity cells in a group of workers exposed to chromic acid and ethylene oxide. In conclusion, our results demonstrated that the occupational exposure to silica in small mines may be a factor that increases the level of DNA damage in the lymphocytes of the workers. The increase in DNA damage in lymphocytes as well as increase in the percentage of MN in buccal cells may indicate a high risk of genotoxicity and possible risk to their health.
Figure 5: Normal control group

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Table 1: Detailed information regarding silica exposure

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Table 2: Assessment of the extent of lymphocyte DNA damage by comet assay and the presence of micronuclei in silica-exposed population (10-15 years exposure)

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  Acknowledgments Top

We are grateful to the Secretary, Ramakrishna Mission Seva Pratishthan, Vivekananda Institute of Medical Sciences, Kolkata, for necessary permission to proceed with the work.

  References Top

1.IARC. Monograph on the Evaluation of Carcinogenic Risks to Humans-Silica. Some Silicates. Coal Dust and Para-Aramide Fibrils. Geneva: IARC, World Health Organization; 1997. p. 211.  Back to cited text no. 1
2.Villarini M, Moretti M, Fatigoni C, Agea E, Dominici L, Mattioli A, et al. Evaluation of primary DNA damage, cytogenetic biomarkers and genetic polymorphisms for CYP1A1 and GSTM1 in road tunnel construction workers. J Toxicol Environ Health A 2008;71:1430-9.  Back to cited text no. 2
3.ICMR BULLETIN. Silicosis-An uncommonly Diagnosed Common Occupational Disease; 1999.  Back to cited text no. 3
4.Anderson D, Yu TW, Dobrzyñska MM, Ribas G, Marcos R. Effects in the Comet assay of storage conditions on human blood. Teratog Carcinog Mutagen 1997;17:115-25.  Back to cited text no. 4
5.Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184-91.  Back to cited text no. 5
6.Palus J, Dziuba³towska E, Rydzyñski K. DNA damage detected by the comet assay in the white blood cells of workers in a wooden furniture plant. Mutat Res 1999;444:61-74.  Back to cited text no. 6
7.Hubber R, Bauchinger M. Development and perspective of the human lymphocyte micronucleus assay. In: Obe G, editor. Advances in Mutagenesis Research. Berlin: Spriger - Verlag; 1990. p. 89-102.  Back to cited text no. 7
8.McKelvey-Martin VJ, Green MH, Schmezer P, Pool-Zobel BL, De Méo MP, Collins A. The single cell gel electrophoresis assay (comet assay): a European review. Mutat Res 1993;288:47-63.  Back to cited text no. 8
9.Fairbairn DW, Olive PL, O'Neill KL. The comet assay: a comprehensive review. Mutat Res 1995;339:37-59.  Back to cited text no. 9
10.Collins A, Dhusinsha M, Franklin M, Somorovska M, Petrovka H, Duthie S, et al. Comet assay in human bio- monitoring studies reliability, validation and applications, Environ. Mol Mutagen 1997;30:95-104.  Back to cited text no. 10
11.Barnes CA, Elsaesser A, Arkusz J, Smok A, Palus J, Lesniak A, et al. Reproducible comet assay of amorphous silica nanoparticles detects no genotoxicity. Nano Lett 2008;8:3069-3074  Back to cited text no. 11
12.Saffiotti U. Silicosis and lung cancer: a fifty-year perspective. Acta Biomedm 2005;2:30-7.  Back to cited text no. 12
13.Wang JJ, Sanderson BJ, Wang H. Cytotoxicity and genotoxicity of ultrafine crystalline SiO2 particulate in cultured human lymphoblastoid cells. Environ Mol Mutagen 2007;48:151-7.  Back to cited text no. 13
14.Yang H, Liu C, Yang D, Zhang H, Xi Z. Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials:the role of particle size, shape and composition. J Appl Toxicol 2009;29:69-78.  Back to cited text no. 14
15.Anreoli C, Leopardi P. Crebetti Detection of DNA damage in human lymphocytes by alkaline single gel electrophoresis after exposure to benzene or benzene metabolites. Mutat Res 1997;377:95-104.  Back to cited text no. 15
16.Vodicka P, Bastlová T, Vodicková L, Peterková K, Lambert B, Hemminki K. Biomarkers of styrene exposure in lamination workers: levels of O6-guanine DNA adducts, DNA strand breaks and mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes. Carcinogenesis 1995;16:1473-81  Back to cited text no. 16
17.Tates AD, van Dam FJ, de Zwart F, Darroudi A, Natarajan T, Rossner K, et al. Biological effects monitoring in industrial workers: from the Czech Republic exposed to low levels of butadine. Toxicology 1996;113:91-9.  Back to cited text no. 17
18.Bolognesi C, Merlo F, Rabboni R, Valerio F, Abbondandolo A. Cytogenetic biomonitoring in traffic police workers: micronucleus test in peripheral blood lymphocytes. Environ Mol Mutagen 1997;30:396-402.  Back to cited text no. 18
19.Karahalil B, Karakaya AE, Burgaz S. The micronucleus assay in exfoliated buccal cells: application to occupational exposure to polycyclic aromatic hydrocarbons. Mutat Res 1999;442:29-35.  Back to cited text no. 19
20.Stich HF, Rosin MP. Quantitating the synergistic effect of smoking and alcohol consumption with the micronucleus test on human buccal mucosa cells. Int J Cancer 1983;31:305-8.  Back to cited text no. 20
21.Kayal JJ, Trivedi AH, Dave BJ, Nair J, Nair UJ, Bhide SV, et al. Incidence of micronuclei in oral mucosa of user of tobacco products singly or various combinations. Mutagenesis 1993;5:31-3.  Back to cited text no. 21
22.Sarto F, Tomanium R, Giacomelli L, Iannini G, Cupiraggi AR. The micronucleus assay in human exfoliated cells of the nose and mouth: application to occupational exposure of chromic acid and ethylene oxide. Mutat Res 1990;244:345-51.  Back to cited text no. 22


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2]

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