coronary artery disease ( cad ) is one of the most common causes of death and disability worldwide ( zaman et al . , 2000 )
. genetic predisposition and interaction with a host of environmental ( endogenous and exogenous ) factors can cause the disease . among the risk factors predisposing to cad are hypertension , obesity , hyperlipidemia , stress , smoking , age , gender and family history ( kasap et al . , 2007 ) .
oxidative stress has also emerged as a major risk factor ( yung et al . , 2006 ) in cad arising through an imbalance between oxidants and antioxidants ( nojiri et al . , 2001 ) .
the oxidative modification of low density lipoproteins ( ldl ) also plays a causative role in the pathogenesis of coronary artery disease ( chisolm and steinberg , 2000 ) .
furthermore , consequences of oxidative stress can cause damage to genomic integrity as atherosclerotic plaques have been reported to exhibit extensive genetic damage ( martinet et al . , 2002 ) .
in fact , reports have also documented the contribution of dna damage to the development and progression of coronary artery disease ( andreassi , 2003 ) .
there has been an increase in heart disease in punjabi population which may be attributed to economic surplus and change in dietary and life - style patterns ( anonymous , 2013 ) .
the changeover from extensive agrarian activities to a sedentary / near sedentary life - style and the adaptation of the western dietary habits across the lower , upper middle and upper socioeconomic status classes , have resulted in increased general and abdominal obesity ( who , 2003 ) . as no studies have come to attention in punjabi populations associating cad with genomic damage , in the present study
, adults ( cad patients and healthy controls ) have been assessed for chromosomal and dna damage in their peripheral blood leukocytes ( pbl ) .
lipid profile , hypertension , diabetes , obesity parameters ( body mass index , waist - hip ratio , waist circumference ) and oxidative stress ( lipid peroxidation , total oxidant status , total antioxidant capacity , oxidative stress index ) . for genetic damage assessment , chromosomal damage in peripheral blood lymphocytes using the cytokinesis - block micronucleus ( cbmn ) assay and dna damage in pbl using the single cell gel electrophoresis ( scge / comet ) assay have been carried out .
the cbmn assay is a well known cytogenetic test - method to assess the genetic effects of spontaneously and mutagenically- induced dna damage ( fenech , 2007 ) and the comet assay is a sensitive biomarker of oxidative stress and dna damage ( balasubramanyam et al . , 2010 ) .
the study proposal was approved by the institutional ethics committee and voluntary written informed consent was taken from all study participants ( n = 65 ) . patients with diagnosed cad were contacted from local hospitals through the cardiologists .
exclusion criteria included cad patients with renal , liver or thyroid disorders , those with negative results on the treadmill test , and those not belonging to the punjabi population .
the control group included healthy adults with no past or present history of heart or any disease and no exposure history , matched for gender , population group and socioeconomic status .
the demographic and disease - related information for each patient was documented on a pre - designed questionnaire .
anthropometric measurements ( height , weight , hip circumference , waist circumference ) were taken following standard methodology ( weiner and lourie , 1981 ) .
derived obesity measures were body mass index ( bmi ) and waist hip ratio ( whr ) . using the criteria of who ( 2004 ) for bmi , the participants were classified as obese or non - obese regarding general obesity , and cut - offs as per snehalatha et al .
( 2003 ) for whr and waist circumference ( wc ) values for abdominal obesity .
physiometric measurements ( using a mercury sphygmomanometer and stethoscope ) were taken after the subject had rested for ten minutes as per the recommendations of american heart association ( kirkendall et al . , 1981 ) .
the average of three measurements were taken as the blood pressure values . the mean arterial blood pressure ( mbp )
was calculated from systolic and diastolic measurements ( prusse et al . , 1989 ) .
venous blood ( 5 ml ) was then drawn from each participant and processed for the cbmn and scge assays .
the cytokinesis - block micronucleus ( cbmn ) assay : - short term lymphocyte cultures were set up ( moorhead et al . , 1960 ) and the cbmn protocol of fenech ( 2000 ) was followed . briefly , the leukocyte - rich plasma ( 2 ml ) was added to 8 ml of rpmi 1640 medium ( himedia , india ) .
the lymphocytes were stimulated to divide by addition of phytohaemagglutinin - m ( 0.5 g / ml pha - m , biological industries , israel ) .
the cultures were incubated for 72 h at 37 c ; after 44 h cytokinesis was blocked by the addition of cytochalasin - b ( 6 g / ml , sigma ) and on completion of incubation of 72 h at 37 c , the cultures were harvested .
following centrifugation ( 800 g , 10 min ) , the pellet was fixed in chilled fixative ( 3 methanol : 1 glacial acetic acid , qualigens , india and srl , india , respectively ) for 30 min .
after centrifugation and two - three washings of the pellet , the cell suspension was used to prepare the slides .
the air - dried slide preparations were stained with 5 % giemsa ( himedia , india ) for 10 - 20 minutes . for each sample ,
a total of 2000 binucleated cells ( 1000/slide ) were scored for the presence of micronuclei ( mn ) and micronucleated cells following the criteria of fenech ( 2007 ) .
random observations by another scorer were done to verify the observations . the nuclear division index ( ndi ) , which is an index of cytostatic effects ,
was calculated ( eastmond and tucker , 1989 ) by using the formula : ndi = ( m1 + 2m2 + 3m3 + 4m4)/n   where m1-m4 represent the number of cells with 1 - 4 nuclei and n is the total number of cells scored . the scge / comet assay : the trypan - blue dye exclusion test for cell viability was performed ( strober , 2001 ) and samples with > 95 % cell viability were processed for the comet assay .
the leukocytic dna damage was ascertained using the alkaline comet assay ( singh et al . , 1998 ) .
in brief , on pre - coated slides with 1 % normal melting point agarose ( nmpa ; srl , india ) , a second layer containing 30 l of blood sample mixed with 110 l of 5 % low melting point agarose ( lmpa , srl , india ) was poured followed by the sandwiching with a third layer of 120 l of 5 % lmpa after the second layer had set .
cells were lysed in freshly prepared cold lysing solution ( 2.5 m nacl , 100 mm na2 edta , 10 mm tris hcl , ph 10.0
; 1 % triton x-100 with 10 % dmso added just before use ) for at least one hour to allow dna unfolding .
then the slides were placed in a horizontal electrophoresis chamber , covered with freshly prepared alkaline electrophoresis buffer ( 0.3 m naoh , 1 mm no2 edta , ph > 13 ) at 4 c for unwinding and expression of alkali - labile sites ( 30 min ) and subsequently electrophoresed ( 25 v , 300 ma , 0.7 - 1.0 -v / cm , 25 min ) .
slide preparations were then washed with neutralizing buffer ( 0.4 m tris hcl ph 7.5 ) to remove alkali and detergents , and left for drying .
the slides were silver stained ( nadin et al . , 2001 ) and coded .
scoring was done using the freely available casp ( comet assay software programme , 3.1.2 version ) .
for this , images were captured with an olympus camera ( e420 , tokyo , japan ) fitted on an olympus microscope ( cx41 , tokyo , japan ) .
a total of 100 cells per sample were scored for per cent tail dna , tail moment and olive tail moment as indices of dna damage .
statistical analysis was performed using the statistical package for the social sciences ( spss , version 16.0 for windows 7 ) .
differences between controls and patients for genetic damage indices were analyzed by the student 's t - test .
demographic variables of patients and control groups were analyzed using the chi - square test .
the association between dna / chromosomal damage and other variables was assessed by the pearson 's correlation test .
the study proposal was approved by the institutional ethics committee and voluntary written informed consent was taken from all study participants ( n = 65 ) . patients with diagnosed cad were contacted from local hospitals through the cardiologists .
exclusion criteria included cad patients with renal , liver or thyroid disorders , those with negative results on the treadmill test , and those not belonging to the punjabi population .
the control group included healthy adults with no past or present history of heart or any disease and no exposure history , matched for gender , population group and socioeconomic status .
the demographic and disease - related information for each patient was documented on a pre - designed questionnaire .
anthropometric measurements ( height , weight , hip circumference , waist circumference ) were taken following standard methodology ( weiner and lourie , 1981 ) .
derived obesity measures were body mass index ( bmi ) and waist hip ratio ( whr ) . using the criteria of who ( 2004 ) for bmi ,
the participants were classified as obese or non - obese regarding general obesity , and cut - offs as per snehalatha et al .
( 2003 ) for whr and waist circumference ( wc ) values for abdominal obesity .
physiometric measurements ( using a mercury sphygmomanometer and stethoscope ) were taken after the subject had rested for ten minutes as per the recommendations of american heart association ( kirkendall et al . , 1981 ) .
the average of three measurements were taken as the blood pressure values . the mean arterial blood pressure ( mbp )
was calculated from systolic and diastolic measurements ( prusse et al . , 1989 ) .
venous blood ( 5 ml ) was then drawn from each participant and processed for the cbmn and scge assays .
the cytokinesis - block micronucleus ( cbmn ) assay : - short term lymphocyte cultures were set up ( moorhead et al . , 1960 ) and the cbmn protocol of fenech ( 2000 ) was followed .
briefly , the leukocyte - rich plasma ( 2 ml ) was added to 8 ml of rpmi 1640 medium ( himedia , india ) .
the lymphocytes were stimulated to divide by addition of phytohaemagglutinin - m ( 0.5 g / ml pha - m , biological industries , israel ) .
the cultures were incubated for 72 h at 37 c ; after 44 h cytokinesis was blocked by the addition of cytochalasin - b ( 6 g / ml , sigma ) and on completion of incubation of 72 h at 37 c , the cultures were harvested .
following centrifugation ( 800 g , 10 min ) , the pellet was fixed in chilled fixative ( 3 methanol : 1 glacial acetic acid , qualigens , india and srl , india , respectively ) for 30 min .
after centrifugation and two - three washings of the pellet , the cell suspension was used to prepare the slides .
the air - dried slide preparations were stained with 5 % giemsa ( himedia , india ) for 10 - 20 minutes . for each sample ,
a total of 2000 binucleated cells ( 1000/slide ) were scored for the presence of micronuclei ( mn ) and micronucleated cells following the criteria of fenech ( 2007 ) .
random observations by another scorer were done to verify the observations . the nuclear division index ( ndi ) , which is an index of cytostatic effects ,
was calculated ( eastmond and tucker , 1989 ) by using the formula : ndi = ( m1 + 2m2 + 3m3 + 4m4)/n   where m1-m4 represent the number of cells with 1 - 4 nuclei and n is the total number of cells scored . the scge / comet assay : the trypan - blue dye exclusion test for cell viability was performed ( strober , 2001 ) and samples with > 95 % cell viability were processed for the comet assay .
the leukocytic dna damage was ascertained using the alkaline comet assay ( singh et al . , 1998 ) . in brief , on pre - coated slides with 1 % normal melting point agarose ( nmpa ; srl , india ) , a second layer containing 30 l of blood sample mixed with 110 l of 5 % low melting point agarose ( lmpa , srl , india ) was poured followed by the sandwiching with a third layer of 120 l of 5 % lmpa after the second layer had set .
cells were lysed in freshly prepared cold lysing solution ( 2.5 m nacl , 100 mm na2 edta , 10 mm tris hcl , ph 10.0
; 1 % triton x-100 with 10 % dmso added just before use ) for at least one hour to allow dna unfolding .
then the slides were placed in a horizontal electrophoresis chamber , covered with freshly prepared alkaline electrophoresis buffer ( 0.3 m naoh , 1 mm no2 edta , ph > 13 ) at 4 c for unwinding and expression of alkali - labile sites ( 30 min ) and subsequently electrophoresed ( 25 v , 300 ma , 0.7 - 1.0 -v / cm , 25 min ) .
slide preparations were then washed with neutralizing buffer ( 0.4 m tris hcl ph 7.5 ) to remove alkali and detergents , and left for drying .
the slides were silver stained ( nadin et al . , 2001 ) and coded .
scoring was done using the freely available casp ( comet assay software programme , 3.1.2 version ) . for this , images were captured with an olympus camera ( e420 , tokyo , japan ) fitted on an olympus microscope ( cx41 , tokyo , japan ) .
a total of 100 cells per sample were scored for per cent tail dna , tail moment and olive tail moment as indices of dna damage .
statistical analysis was performed using the statistical package for the social sciences ( spss , version 16.0 for windows 7 ) .
differences between controls and patients for genetic damage indices were analyzed by the student 's t - test .
demographic variables of patients and control groups were analyzed using the chi - square test .
the association between dna / chromosomal damage and other variables was assessed by the pearson 's correlation test .
the demographic and clinical characteristics of patients and controls are presented in table 1(tab . 1 ) .
cad patients had been diagnosed by the cardiologists on the basis of clinical symptoms , ecg findings , treadmill test and echocardiography .
there were 26 males ( mean age 49.62  1.73 y ) and 20 females ( mean age 57.30  1.31 y ) in the patient group .
the control group included 13 males ( mean age 53.54  1.17 y ) and 6 females ( mean age 55.67  1.33 y ) .
on the basis of bmi , there were 14 obese patients ( bmi  25.0 kg / m ) while the whr revealed that 26 male and 20 female patients were obese as per the cut - off values given by snehalatha et al .
( 2003 ) . on the basis of the waist circumference cut - offs ( snehalatha et al . , 2003 ) , 23 male ( > 85 cm ) and 19 female ( > 80 cm ) patients had abdominal obesity .
alcohol drinking was observed in 52.20 % male patients and in 31.60 % male healthy controls .
non - vegetarian diet was preferred by 58.70 % of the patients and 52.60 % of the controls and sedentary life style was observed by 71.70 % cad patients and only by 36.80 % of controls . regarding medication in the patients group
, 67.40 % were taking aspirin ( anti - inflammatory ) , 58.70 % lasix ( diuretic ) , 52.20 % metoprolol ( beta - blocker ) and 47.80 % were taking ramipril ( ace - inhibitor ) . socioeconomic status ( ses )
there were almost equal percentage of patients ( 15.20 % ) and controls ( 15.80 % ) belonging to upper ses .
similarly , 76.10 % patients and 13.70 % controls belonged to upper - middle ses , while 8.70 % among patients and 10.50 % among controls had lower - middle ses .
information about the history of disease revealed that date - of - onset / diagnosis ranged from 35 - 72 y ( average 53.09  1.3 y ) and the patients were on medication from 1 - 15 y ( average 5.04  0.6 y ) . on chi - square analysis it was observed that the demographic variables of patients and controls matched for age , gender , alcohol drinking , diet preference , socioeconomic status , whr ( men ) and waist circumference ( women ) , while bmi , diabetes , dyslipidemia , physical activity , whr ( women ) , waist circumference ( men ) , blood pressure , mbp and medication showed differences being characteristic of patients . in table 2(tab .
2 ) , the information on genetic damage indices in patients and controls is presented .
the trypan - blue dye exclusion test had revealed 95.60 - 99.80 % cell viability of all samples .
the comet parameters used as metrics of dna damage were tail moment ( tm = tail dna% x tail length in arbitrary units ,
au ) , tail dna% ( td = amount of dna in comet tail in percentage ) and olive tail moment ( otm = tail mean - head mean x % dna in tail in au ) .
a comparison between patients and controls for these dna damage indices revealed that the values were significantly ( p < 0.001 ) higher in patients compared to those in controls .
however no gender differences for these values were observed among patients and hence the respective data were pooled for genders in both , the control and patient groups .
separate analysis also showed highly significant differences ( p < 0.001 ) for both , male and female patients from their control counterparts for dna damage indices .
for the assessment of chromosomal damage , the binucleated cells with micronuclei ( bnmn ) , number of micronuclei in binucleated cells ( mn ) and the nuclear division index ( ndi ) were studied .
statistical analysis revealed that the bnmn and mn values were significantly ( p < 0.001 ) higher and ndi values lower in patients than in controls .
as observed for dna damage , no gender differences for chromosomal damage were observed among patients and controls , though values in male and female patients for bnmn and mn were significantly higher ( p < 0.001 ) in patients while ndi values were significantly ( p < 0.001 ) higher in controls . on correlation and association analyses ( table 3(tab .
3 ) ) , significant positive correlation ( correlation coefficient = r ) was observed between bnmn and diabetes mellitus ( r = 0.297 , p = 0.045 ) and between ndi and mda levels ( r = 0.296 , p = 0.045 ) and with age ( r = 0.571 , p = 0.000 ) .
positive correlation was observed between tail dna % and diabetes mellitus ( r = 0.442 , p = 0.004 ) and with whr ( r = 0.349 , p = 0.017 ) and between tail moment and age ( r = 0.312 , p = 0.035 ) . on multiple linear regression analysis ( table 4(tab .
4 ) ) , regression coefficient ( b ) values for age ( b = 0.076 , p = 0.035 ) , whr ( b = 25.859 , p = 0.018 ) and diabetes mellitus ( b = 2.251 , p = 0.004 ) were observed to be independent predictors for dna damage and ndi for chromosomal damage ( b = 0.007 , p = 0.001 ) . in order to discern
if there was any association between dna damage and chromosomal damage parameters , correlation analysis was carried out but it revealed no significant associations ( table 5(tab .
5 ) ) . as oxidative stress and lipid profile biomarkers of this study group had earlier been reported to be significantly higher among the patients ( bhat et al . , 2012 ) , it was also thought appropriate to ascertain whether there was any correlation ( table 3(tab .
4 ) ) of these variables with genetic damage . correlation and regression analyses were hence performed for genetic damage ( dna / chromosomal damage ) with oxidative stress parameters of malondialdehyde ( mda ) , total oxidant status ( tos ) , total antioxidant status ( tas ) and oxidative stress index ( osi ) and also with lipid profile biomarkers ( total cholesterol , high density lipoprotein cholesterol ( hdl - c ) , triglycerides , very low density lipoprotein ( vldl ) and low density lipoprotein ( ldl ) .
these showed neither correlation nor association with genetic damage except hdl - c and mda levels which showed positive correlation ( r = 0.322 , p = 0.029 ; r = 0.296 , p = 0.046 respectively ) and regression ( b = 0.259 , p = 0.029 ; b = 0.013 , p = 0.046 respectively ) with per cent tail dna and ndi respectively .
it has often been reported that genetic damage may be caused by both , extrinsic and intrinsic environments ( gray and bennett , 2011 ) .
the dna damage can manifest as dna strand - breaks , alkali - labile sites , incomplete repair sites and cross - links while chromosomal damage appears as chromosomal aberrations ( gaps , breaks , translocations , rings , insertions ) as well as micronuclei ( mn ) .
mn originate from chromosome fragments or whole chromosomes that are not included in the main daughter nuclei during nuclear division ( fenech , 2000 ) . in the present study ,
the scge assay was used to measure the level of dna damage ( unrepaired ) and the cbmn assay to measure the level of chromosomal damage ( repaired ) in patients and controls .
the results from the comet assay demonstrated that patients diagnosed with cad had higher values of percentage of tail dna ( 2.1x ) , tail moment ( 3 x ) and olive tail moment ( 3 x ) , indicative of higher dna damage in peripheral blood leukocytes of cad patients than the normal controls .
there were no gender differences for these indices on statistical analysis implying that in this study group , genetic damage levels ( both dna and chromosomal ) are similar in males and females among patients and controls . the results of the present study have shown significant increase in unrepaired and repairable genetic damage in peripheral blood leukocytes / lymphocytes of patients with coronary artery disease .
reports in literature have implicated excessive oxidative stress and inadequate defenses in the pathogenesis of cardiovascular disease ( andreassi , 2003 ) besides genetic damage in atherosclerotic plaques ( martinet et al . ,
2002 ) . as oxidative stress has been implicated as a major contributor in the progression of atherosclerosis ( norman et al . , 2001 )
, it could also be a causative factor for dna damage in coronary artery disease as dna damage occurs frequently in cells exposed to reactive oxygen species ( botto et al . ,
the cad patients of the present study also had significantly increased ( 3 folds ) oxidative stress as given in an earlier work ( bhat et al . , 2012 ) .
this oxidative stress hence could be contributing to the observed dna and chromosomal damage in cad patients .
as dyslipidemia is an important causative factor for coronary artery disease ( arca et al . , 2007 ) , the patients of the present study had earlier been assessed for their lipid profile and had higher levels of triglycerides and total cholesterol and low levels of high density lipoprotein - cholesterol despite treatment ( bhat et al . , 2012 ) .
the hdl - c and mda levels showed positive correlation with genetic damage on correlation and regression analyses implying that dyslipidemia also induced oxidative stress ( higher mda levels ) and could also be contributing to the observed genetic damage in these patients . in literature , various studies have demonstrated increased genetic damage in patients with heart disease .
increased production of oxidant free radicals was observed in patients with ischemic heart disease ( logacheva et al . , 2001 ) and with evidence of oxidative dna damage in cells within the atherosclerotic plaque ( mahmoudi et al . ,
2006 ) implying that genomic damage may itself be inducing cad . in their study on indian cad patients , rajesh et al .
( 2011 ) reported increased dna damage ( per cent tail dna ) while botto et al .
( 2001 ) had also reported increased dna damage levels in patients with coronary artery disease .
( 2005 ) had also documented that dna strands breaks , oxidized pyrimidines and altered purines were significantly higher in leukocytes of cad patients .
the key advantage of the mn assay lies in its ability to detect both , clastogenic and aneugenic events , and to identify cells which have divided once in culture ( fenech , 2002 ) .
the results of the present study showed that bnmn ( 1.5 fold ) and mn ( 1.6 fold ) values were significant increased in patients while ndi ( a biomarker of cytotoxicity ) was significantly higher in controls ( p < 0.001 ) than in patients implying that the cell proliferation was reduced while genomic damage was elevated in patients .
increase in bnmn among cad patients in an italian population had also been earlier reported ( andreassi et al . , 2005 ) .
the results of the present study are also supported by those of botto et al .
( 2001 ) who had observed approximately 5.6 folds increase in mn levels in cad patients while andreassi et al .
the observed dna damage in patients of the present study could also have been contributed by the action of the medications prescribed to these patients namely , asprin ( anti - inflammatory ) , lasix / furosemide ( diuretic ) , metoprolol ( beta - blocker ) and ramipril ( ace - inhibitors ) .
studies on genotoxicity of asprin have shown positive effects in bacillus subtilis assay ( kawachi et al . , 1980 ) and invitro in the chinese hamster lung cells ( ishidate , 1983 ) though negative effects have also been reported in the ames test ( mccann et al . , 1975 ) and in the chinese hamster ovary cells ( stich et al . , 1981
( 2012 ) has reported dna strand breaks in mice hepatocytes detected by comet assay .
chromosomal aberrations in chinese hamster ovary and lung fibroblast cells and in mouse germ cells have also been reported ( jameela et al . , 1979 ; bucher , 1989 ) , yet the genotoxic effects of furosemide are not well characterized ( bucher , 1989 ; brambilla et al . , 2006 ) .
recently , on performing mutagenecity testing ( ames test , comet assay ) the effects of degradation products of metoprolol revealed positive genotoxicity ( sojic et al .
. therefore the genetic - damaging effects of these drugs can not be ruled out .
furthermore , the carcinogenicity of cardiovascular drugs , including diuretics , beta blockers , calcium antagonistics and ace - inhibitors has also been rebated upon ( grossman et al . , 1999 ) and requires further research .  
the significantly increased dna and chromosomal damage observed in the pbl of cad patients of the present study is of concern as genetic damage is an early indicator of malignancy ( andreassi et al .
, 2007 ; manikantan et al . , 2010 ) and can also promote ageing and neurodegenerative diseases ( farooqui and farooqui , 2009 ; muralidhar et al . , 2012 ) .
hence there is a need for preventive management for these aspects in patients with coronary artery disease .
michael fenech ( csiro human nutrition , adelaide , australia ) for providing cytochalasin - b . the present study was carried out with support out of the departmental ugc - sap grant .
 dna and chromosomal damage in peripheral blood leukocytes of patients with coronary artery disease ( cad ) were investigated by using the single cell gel electrophoresis assay /comet and cytokinesis- block micronucleus ( cbmn ) assays , respectively . 
 the case - control study comprised patients with cad ( n = 46 ; average age 53.0  1.27 y ) undergoing treatment at local hospitals , and healthy age - and sex - matched controls ( n = 19 ; average age 54.21  0.91 y ) from the general population . 
 the results of the comet assay revealed that the mean values of dna damage were significantly ( p < 0.001 ) higher in cad patients than in controls ( tail dna% 11.55  0.38 vs. 5.31  0.44 ; tail moment 6.17  0.31 vs. 2.93  0.21 au ; olive tail moment 3.52  0.23 vs. 1.25  0.11 au ) . 
 the mean values of chromosomal damage were also significantly higher ( p < 0.001 ) in cad patients than in controls ( binucleated cells with mn- 28.15  1.18 vs. 18.16  2.59 ; micronuclei 29.52  1.21 vs. 18.68  2.64 , respectively ) while nuclear division index ( 1.48  0.01 vs. 1.63  0.01 ) was significantly higher ( p < 0.001 ) in controls . the results of the present study indicate that coronary artery disease patients had increased levels of both , unrepaired ( dna ) and repaired ( chromosomal ) genetic damage which may be a pathological consequence of the disease and/or the drug - treatment . 
 this accumulation of dna / chromosomal damage is of concern as it can lead to the development of cancer with increased chances of morbidity and mortality in the cad patients . 
Introduction
Materials and Methods
Subjects
Data and sample collection
Statistical analysis
Results
Discussion
Acknowledgement