asbestosis is one of the most frequently studied diseases associated with the inhalation of asbestos fibers . the exposure to asbestos is known to cause inflammation , fibrous scarring in the lung , and cancer [ 1 , 2 ] . it has been suggested that , in addition to reactive oxygen species ( ros ) , the reactive nitric species ( rns ) , such as nitric oxide ( no ) , may also be involved in the development of asbestos - related diseases [ 36 ] . nitric oxide is a free radical which readily reacts with ros , such as superoxide anion , producing peroxynitrite , which can generate hydroxyl radicals [ 711 ] . in the living organisms no is synthesized by nitric oxide synthases ( noss ) , which catalyse the conversion of amino acid l - arginine and molecular oxygen into no and l - citrulline [ 11 , 12 ] . three isoforms of nos have been identified : neuronal nos ( nnos or nos1 ) , inducible nos ( inos or nos2 ) , and endothelial nos ( enos or nos3 ) . inducible nos is often present at sites of chronic inflammation [ 5 , 11 ] . nitric oxide generation is thought to be an important mediator of cell injury and inflammation caused by asbestos . studies indicate that asbestos fibers may upregulate the activity of inos and thus the production of no by alveolar macrophages and pulmonary epithelial cells [ 5 , 6 , 1315 ] . several studies described the association between no production and the adverse effects of asbestos [ 36 , 13 ] . reported an association between the magnitude of no production by lung epithelial cells and dna oxidation at in vitro exposure to asbestos . treatment of human lung epithelial cells with asbestos was shown to result in the synthesis of inos mrna and increased intracellular levels of nitrate , a stable oxidation product of no . in addition , a temporal correlation was demonstrated between no production by alveolar macrophages and neutrophil infiltration into lung after inhalation of asbestos . the findings of these studies suggest that no may play an important role in the initiation and progression of asbestosis . however , the studies in inos knockout mice indicate that inos - derived no exerts a dual role in acute asbestos - induced lung injury as inos deficiency resulted in an exacerbated inflammatory response but improved oxidant - promoted lung tissue damage . several types of polymorphisms have been identified in the promoter region of the inos gene , among them the tandem repeat number polymorphism of a ccttt pentanucleotide ( ( ccttt)n ) [ 17 , 18 ] . the ccttt pentanucleotide repeats within the promotor region of the human inos gene have been associated with the biochemical promoter activity . several studies have suggested an association between inflammatory diseases or cancer and the number of the ccttt repeats [ 1821 ] . however , to our knowledge and according to the literature available , this polymorphism has not been studied so far in association with asbestosis .   in this study we investigated whether inos ( ccttt)n polymorphism is associated with an increased risk of asbestosis in workers exposed to asbestos . the study population comprised 262 subjects who were diagnosed with asbestosis as an occupational disease and 265 subjects with no asbestos disease as the control group . all the subjects were selected from a cohort of 2080 workers who were occupationally exposed to asbestos in the asbestos cement manufacturing plant of salonit anhovo , slovenia , and presented at the state board for recognition of occupational asbestos diseases in the period between 1 january 1998 and 31 december 2003 .   to obtain the information about smoking , all cases and controls were interviewed by the same investigator , using a standardized questionnaire [ 23 , 24 ] . for each subject included in the present study , the cumulative asbestos exposure data were obtained from the previous study . the diagnosis of asbestosis or  no asbestos - related disease  was confirmed by two groups of experienced occupational experts of the state board for recognition of occupational asbestos diseases at the clinical institute of occupational medicine . each group of experts consisted of an occupational physician , a radiologist , and a pulmonologist skilled in diagnosis of asbestos - related diseases . the diagnosis was based on the helsinki criteria for diagnosis and attribution of asbestos diseases   and on the american thoracic society recommendations . high - resolution computer tomography ( hrct ) was used for radiological diagnosis of asbestosis . in genotyping of the ( ccttt)n in the inos gene promoter was performed by the amplification of the corresponding region of the promoter by pcr using a primer pair with one primer being fluorescently labelled . the amplified fragments were analysed on the abi prism 310 genetic analyser ( applied biosystems ) , and the number of polymorphic repeats was determined with genemapper analysis software ( applied biosystems , norwalk , usa ) as previously reported . first , the statistical analysis included the descriptive statistics , t - test , and  test . next , univariate logistic regression was performed to estimate the risk of asbestosis in relation to individual variables ( inos , cumulative asbestos exposure , gender , age , and smoking ) . the subjects with asbestosis were on average 61 and the controls 57 years old ( t = 5.18 , p = .000 ) . comparison of smoking between the cases and the controls showed no statistically significant difference (  = 0.01 , p = .919 ) . approximately forty - five percent of subjects ( 117 cases and 120 controls ) were ever smokers . the average cumulative asbestos exposure was significantly higher in subjects with asbestosis compared to those without asbestos - related disease ( 37.67 fibres / cm - years for cases and 11.23 fibres / cm - years for controls ; = 4.78 , p = .000 ) . genotyping of the ( ccttt)n in the inos gene promoter was successful in 258 cases and 259 controls . thirteen alleles for the inos microsatellite ranging from 166 to 226 base pears ( bp ) ( 6 to 18 ccttt repeats ) were observed ( table 1 ) . the alleles with 11 ccttt repeats were defined as short alleles ( s ) and alleles with 12 repeats as long alleles ( l ) ( table 1 ) . accordingly , the subjects were classified into three groups of genotypes : ss , sl , and ll . inducible nitric oxide synthase ll genotype was observed in 132 cases and 121 controls , sl genotype in 83 cases and 96 controls , while ss genotype was found in 43 cases and 42 controls ( table 2 ) . when combining genotypes , no significant difference was found in the frequency of the inos ll genotype versus the frequency of combined sl and ss genotypes ( table 2 ) . no association has been observed between asbestosis and smoking ( ever / never ) ( or = 0.98 , 95% ci = 0.691.39 ) . cumulative asbestos exposure was logarithmically transformed , leading to an or of asbestosis of 3.21 ( 95% ci  = 2.434.23 ) . determining the associations between inos genotypes and asbestosis , the or of asbestosis was 1.07 ( 95% ci = 0.651.74 ) for the ll genotype versus ss genotype , 1.26 ( 95% ci = 0.861.85 ) for the ll genotype compared to sl genotype , 0.84 ( 95% ci = 0.501.42 ) for the sl genotype versus ss genotype , and 1.20 ( 95% ci = 0.851.69 ) for the ll genotype compared to the combined sl and ss genotypes . the risk of asbestosis for the inos ll genotype versus the combined sl and ss genotypes remained practically unchanged after adjustment by gender , age , smoking , and cumulative asbestos exposure ( table 3 ) . it has been suggested that inos - derived no may play an important role in the development of asbestos - related diseases [ 5 , 6 , 1315 ] . in a nested case - control study the authors investigated the influence of the inos ( ccttt)n polymorphism on the risk of developing asbestosis . a slightly ( 20% ) higher risk of asbestosis for the inos ll genotype compared to the combined sl and ss genotypes and almost 30% for the ll versus sl genotype has been detected . this finding , although borderline significant , could be in agreement with the observations of some other studies which demonstrated an association between a disease and longer forms of ( ccttt)n repeat in the inos promoter region [ 18 , 20 , 21 ] . these results could be explained by a higher promoter activity of the inos gene which has been shown to increase with the ( ccttt)n repeat number in an in vitro study   and consequently increased production of no . since no is a free radical and its production has been associated with pulmonary damage , inflammation , and disease progression caused by asbestos [ 5 , 6 ] , our finding could be considered as biologically plausible . all the cases and controls who participated in this study were recruited in a small geographical area with an ethnically homogenous population . therefore , no bias was introduced by genetic heterogeneity .   although the board of experts used the hrct as a widely recommended method for the diagnosis of asbestosis [ 25 , 26 ] in all subjects , there is still a slight possibility that some subjects might have been recruited as controls when they had already developed asbestosis , which could be diagnosed only by the histopathological examination . in that case considering that our study included cases who had inos ll genotype more frequently than the controls , the association between asbestosis and inos ll genotype might have been slightly stronger in that case . in conclusion , to our knowledge , this is the first study investigating the association between asbestosis and inos genetic polymorphism . considering that the results of the current study are borderline significant , further studies including a larger number of subjects are needed to clarify this association .