crystalline silica has been classified as a human carcinogen by international agency for research on cancer . 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 . 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 . clearance for the study was obtained from the ethics committee of the vivekananda institute of medical sciences . ( i ) mine and quarry workers with a history of 1015 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 . 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 4c to our laboratory on the same day and stored overnight at 4c . there is no loss of cell viability at 4c or room temperature up to 8 days according to anderson et al . 20 l 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 . the rest of the blood was used for routine analysis such as hemoglobin ( hb ) concentration , packed cell volume ( pcv ) , etc . 110 l 0.6% normal melting agarose ( nma ) was coated on a fully frosted slide for a sound attachment and solidified for 5 min . 0.6% low melting point agarose mixed with 10 l lymphocyte was coated over the first layer . a third , agarose , layer ( 75 l ) was coated over the second lymphocyte agarose layer at 4c for another 10 min . after removal of the cover glass , the slides were immersed in the lysing solution ( 2.5 mol . nacl , 100 mm 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 , 4c ) to a level of approximately 0.25 cm above the slide . the slides were allowed to set in this high ph buffer for 2025 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 . 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 515560 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 . 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 . slides were stained in giemsa stain and 1000 cells were scored by the same person . photograph of dna low - level damage photograph of dna medium - level damage photograph of dna high - level damage photograph of dna complete damage 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 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 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 . 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.[56810 ] 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 . in vitro toxicity testing by the comet assay has been demonstrated to be quantitatively reproducible . much work has been done on the association of silicosis with lung cancer and it was found that ultrafine silicon dioxide is cytotoxic and genotoxic in cultured human cells , while yang et al . 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 and in workers in a plastic factory , whereas no statistically significant differences were observed in a study of workers exposed to 1 , 3-butadiene . the increase in micronuclei ( mn ) bolognesi et al . found higher frequency of mn in traffic police officers exposed to lead , while villarini et al . 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 . reported that age , sex , and smoking habit did not influence the mn frequency . stich and rosin 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 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 . detailed information regarding silica exposure assessment of the extent of lymphocyte dna damage by comet assay and the presence of micronuclei in silica - exposed population ( 1015 years exposure )
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 .