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

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.

INTRODUCTION

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 (SiO2) 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.[23]

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

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

Figure 2

Photograph of DNA medium-level damage

Figure 3

Photograph of DNA high-level damage

Figure 4

Photograph of DNA complete damage

RESULTS AND DISCUSSION

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.[568-10] 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.[910] 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[1213] 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[2122] 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.

Table 1

Detailed information regarding silica exposure

Figure 5

Normal control group

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)