
 study population - in total , 49 patients with chronic viral hepatitis , 
 consisting of 28 hbv - infected patients and 21 hcv - infected patients , and 33 healthy , 
 non - infected controls were included in the study .
the patients were recruited from the 
 clinic of infectious diseases of julio muller hospital ( federal university of mato 
 grosso , cuiab , state of mato grosso , central - west brazil ) .
chronic hbv infection was 
 confirmed by persistent hbv surface antigenemia lasting more than six months .
chronic 
 hcv infection was confirmed by the presence of hcv rna in blood tests .
cirrhosis was 
 diagnosed by liver biopsy or based on clinical observations , laboratory tests or 
 ultrasonographic evidence .
non - infected subjects were recruited from a group of healthy 
 blood donors at the public blood bank of mato grosso state .
information about alcohol 
 consumption , tobacco use , ethnicity and age was obtained from medical records and from 
 an interviewer - administered questionnaire , which also included questions about exposure 
 to mutagens and any history of cancer in the individual . 

cytokinesis - block mns ( cbmn ) assay - the cbmn assay was performed as 
 described by fenech and morley ( 1985 ) , with 
 minor modifications . in total , 5 l of venous blood was collected in heparin - vacutainer 
 tubes ( becton & dickinson , franklin lakes , nj , usa ) and lymphocyte cultures ( 2 per 
 subject ) were established using 0.3 ml of whole blood added to rpmi-1640 medium 
 ( sigma - aldrich , st .
louis , mo , usa ) supplemented with 20% foetal calf serum ( cultilab , 
 campinas , sp , brazil ) , 0.001% penicillin ( vetec , duque de caxias , rj , brazil ) , 0.0005% 
 streptomycin ( sigma - aldrich ) and 2% phytohemagglutinin ( cultilab ) . after the cultures 
 were incubated for 44 h at 37c in a bod incubator ( eletrolab , so paulo , sp , brazil ) , 
 cytochalasin b ( sigma - aldrich ) was added to the cultures ( 6 g / ml ) .
the cells were 
 harvested by centrifugation at 72 h after the culture was initiated .
the lymphocytes 
 were treated with a hypotonic solution ( 1% sodium citrate w / v ) and fixed in a solution 
 of methanol : acetic acid ( 3:1 v / v ) ; in both cases , the solutions were ice cold and 
 freshly prepared .
the cell suspension was dropped onto a pre - cooled microscope slide and 
 air dried before being stained for 5 - 7 min with 5% giemsa in sorensen phosphate buffer 
 ( 0.06 m na 2 hpo 4 and 0.06 m kh 2 po 4 , ph 
 6.8 ) .
microscopic analysis was performed with a light microscope ( nikon , melville , ny , 
 usa ) at 400x magnification . for each individual
, 2,000 binucleated cells were analysed 
 for the presence of mn , npbs and nbuds in accordance with previously established 
 criteria ( fenech et al .
we calculated 
 the frequency of each biomarker ( number in 1,000 ,  ) using the following formula :  of x 
 = ( number of x/2,000 ) x 1,000 , where x is mn , npbs or nbuds ( montero et al .
mutagen sensitivity evaluation - to determine mutagen sensitivity , the 
 well - established mutagen dxr was used to treat lymphocytes from patients and 
 non - infected subjects at 44 h after the culture was initiated .
the cells were treated 
 with 0.15 g / ml dxr ( bergamo , taboo da serra , sp , brazil ) diluted in sterile distilled 
 water for 28 h , which brought the total culture time to 72 h. the experimental 
 conditions for the dxr treatment were previously established in preliminary experiments . 

the conditions for cell harvesting and slide preparation were described in the previous 
 section .
sensitivity to dxr was expressed as induced dna damage : [ ( mean mn after 
 dxr)-(basal mean mn ) ] . 

statistical analysis - an age comparison between groups was performed 
 using one - way anova and the bonferroni post - hoc test .
when the data exhibited unequal 
 variance , the median numbers of mn , npbs and nbuds were compared between the groups 
 using the non - parametric kruskal - wallis test followed by dunn s post - hoc test . 

similarly , the median numbers of mn obtained in the mutagen sensitivity test were 
 compared between the groups using the mann - whitney u test .
the mean 
 measurements of the induced dna damage were compared using the student s 
 t test .
the g - test or the    test was used to compare 
 ethnicity , sex , alcohol intake and tobacco intake between the groups .
a linear 
 regression model was constructed using the stata 8.2 software programme ( statacorp , 
 college station , tx , usa ) to verify the independence of the frequencies of mn , npbs and 
 nbuds from the virus type , sex , age , alcohol intake , tobacco intake and the use of 
 antiviral drugs .
the 
 statistical analyses were performed using the statistical software programme bioestat 
 5.0 ( ayres et al .
ethics - this study was approved by the ethical research board of julio 
 muller hospital ( protocol 439/cep - hujm/07 ) and informed consent to voluntarily 
 participate was given by all of the subjects .
of the 49 patients included in the study , 28 were chronically infected with hbv ( 21 
 males and 7 females ) and had a mean age of 36.1 years ( ranging from 21 - 56 years ) , while 
 21 were chronically infected with hcv ( 13 males and 8 females ) and had a mean age of 
 44.1 years ( ranging from 27 - 55 years ) .
the non - infected control group consisted of 23 
 males and 10 females with a mean age of 37.4 years ( ranging from 20 - 52 years ) .
no 
 differences were observed between the non - infected group and the hbv or hcv - infected 
 patients with regard to their sex ( p = 0.97 ) and smoking habits ( p = 0.16 ) .
the mean age 
 was similar between the infected patients and the non - infected control subjects ; 
 however , the mean age of the hcv - infected patients was significantly higher than that of 
 either the chronically hbv - infected group or the controls ( p < 0.05 ) .
alcohol 
 consumption among the study subjects ranged from 0 - 8 g / day and was more frequently 
 reported by the non - infected control subjects ( 45.45% ) than by the hbv - infected patients 
 ( 17.86% ) or the hcv - infected patients ( 14.28% ) ( p = 0.003 ) .
cirrhosis was present in 
 eight hbv - infected patients ( 28.6% ) and three hcv - infected patients ( 14.29% ) ( p = 0.31 ) 
 ( table i ) .
seven patients ( 6 hbv - infected and 2 hcv - infected patients ) were undergoing 
 antiviral therapy at the time of the study ( 1 lamivudine and tenofovir , 2 ribavirin and 
 -interferon and 3 tenofovir ) . 

table idemographic and clinical characteristics of patients chronically infected 
 with hepatitis b virus ( hbv ) or hepatitis c virus ( hcv ) and of control 
 subjectsdemographic and clinical parameterscontrols ( n = 33 ) n ( % ) total patients ( n = 49 ) n ( % ) hbv ( n = 28 ) n ( % ) hcv ( n = 21 ) n ( % ) 

 males23 ( 69.7)36 ( 69.4)21 ( 75)13 ( 61.9)females10 ( 30.3)15 ( 30.6)7 ( 25)8 ( 38.1)age ( years ) range37.4  10.4 ( 20 - 52)37.4  10.3 ( 20 - 55)36.1  10.36   ( 27 - 55)37.41  7.7 ( 20 - 52)smoking habit1 ( 3.03)8 ( 16.3)4 ( 14.3)4 ( 19.04)alcohol consumption15 ( 45.4 ) 
 8 ( 16.3)5 ( 17.9)3 ( 14.3)cirrhosis-11 ( 22.4)8 ( 28.6)3 ( 14.3 ) 
 a : anova and bonferroni post - hoc test , p < 0.05 ; 
 b : g test , p = 0.004 . 
 
 a : anova and bonferroni post - hoc test , p < 0.05 ; 
 b : g test , p = 0.004 . the numbers of mn , npbs and nbuds observed in the patient group were 7.01  3.23 , 2.76  
 2.08 and 4.57  2.98 , respectively .
the frequencies of mn and npbs , but not nbuds , were 
 significantly increased ( p < 0.0001 ) compared with the frequencies in the controls 
 ( 4.41  2.15 and 1.15  0.97 for mn and npbs , respectively , in the controls ) . 

considering the hbv - infected patients and the hcv - infected patients separately , the 
 numbers of mn in the hbv - infected patients ( 7.18  3.57 ) and hcv - infected patients ( 6.78 
  2.80 ) were significantly higher than in the non - infected group ( 4.41  2.15 ) ( p < 
 0.0001 ) . the numbers of npbs and nbuds in the hbv - infected patients ( 3.27  2.40 and 
 4.71  2.79 , respectively ) and in patients infected with hcv ( 2.09  1.33 and 4.38  
 3.28 , respectively ) were increased relative to those of the control group ( 1.15  0.97 
 and 2.98  1.31 , respectively ) .
however , only the difference between the hbv - infected 
 group and the control group was statistically significant ( p = 0.03 ) ( table ii ) .
the results were independent of age , 
 sex , alcohol consumption , tobacco intake and the presence of cirrhosis for mn ( r = 0.22 , 
 p = 0.02 ) , npbs ( r = 0.23 , p < 0.001 ) and nbuds ( r = 0.15 , p = 0.03 ) . the antiviral 
 therapy did not significantly influence any of the analysed parameters .
the frequency of 
 mn was significantly influenced by sex only for males aged < 40 years in the control 
 group , who showed a significantly lower frequency of mn ( 3.46  1.12 ) than did females 
 ( 5.63  1.60 ) of the same age ( p = 0.01 ) . the frequency of mn and npbs was significantly 
 higher in males , among all patients and within the hbv - infected group in comparison with 
 the controls ( p < 0.01 ) ( table iii ) .
the 
 frequency of npbs was significantly higher in females aged > 40 years in comparison 
 with the controls ( p < 0.01 ) .
the frequency of mn , npbs and nbuds was not 
 significantly higher in male patients with cirrhosis in comparison with male patients 
 without cirrhosis ( table iv ) . 

table iifrequency of micronucleus ( mn ) , nucleoplasmatic bridges ( npb ) and nuclear 
 buds ( nbuds ) in hepatitis b virus ( hbv ) or hepatitis c virus ( hcv)-infected 
 patients and controlsgroupsmn ( mean  sd)npb ( mean  sd)nbuds ( mean  sd ) 

 controls ( n = 33)4.41  2.151.15  0.972.98
 1.31total patients ( n = 49)7.01  3.23 
 2.76  2.08 
 4.57  2.98hbv ( n = 28)7.18  3.57 
 3.27  2.40 
 4.71  2.79 
 hcv (
n = 21)6.78  2.80 
 2.09  1.334.38  3.28 
 a , b : statistically significant in 
 comparison to the controls ( a : p < 0.0001 ; 
 b : p = 0.03 ; kruskal - wallis test with dunn s post - hoc 
 test ) ; sd : standard deviation .. 
 
 a , b : statistically significant in 
 comparison to the controls ( a : p < 0.0001 ; 
 b : p = 0.03 ; kruskal - wallis test with dunn s post - hoc 
 test ) ; sd : standard deviation .. 
 table iiithe effect of age and sex on frequency of micronucleus ( mn ) , 
 nucleoplasmatic bridges ( npb ) and nuclear buds ( nbuds ) n hepatitis b virus 
 ( hbv ) or hepatitis c virus ( hcv)-infected patients and controlsgroupsmn ( mean  sd)npb ( mean  sd)nbuds ( mean  sd ) 

 controls ( n = 33 )  males < 40 years ( n = 14)3.46  1.12 
 1.32  1.312.86  2.67  males  40 years ( n = 9)4.06  1.590.83  0.52.67  1.17  females < 40 years ( n = 4)5.63  1.601.50  0.824.13  0.85  females  40 years ( n = 6)6.33  3.541.0  0.633.0
 1.26total patients ( n = 49 )  males < 40 years ( n = 14)6.79  4.49 
 3.5  2.97 
 4.03  2.45  males  40 years ( n = 20)7.25  2.92 
 3.08  1.45 
 5.10  3.72  females < 40 years ( n = 6)6.92  2.853.0  2.344.0  2.61  females  40 years ( n = 9)6.89  2.103.50  1.44 
 4.61  2.25hbv ( n = 28 )  males < 40 years ( n = 12)6.71  4.82 
 3.27  3.14 
 4.42  2.45  males  40 years ( n = 9)7.33  2.03 
 3.27  1.51 
 5.89  3.46  females < 40 years ( n = 4)7.0  3.633.25  2.963.25  2.90  females  40 years ( n = 3 ) 
 8.83  1.533.12  0.484.33  0.76hcv ( n = 21 )  males < 40 years ( n = 2)7.25  2.475.0  01.75  0.35  males  40 years ( n = 11)7.18  3.59 
 2.67  1.37 
 4.45  3.97  females < 40 years ( n = 2)6.75  1.062.50  0.715.50  1.41  females  40 years ( n = 6)5.92  1.663.80  1.92 
 4.75  2.79 
 a : statistically significant in comparison to females of the 
 same age - class ( p = 0.01 , mann - whitney u test ) ; 
 b : statistically significant in relationship to controls 
 of the same age class ( p < 0.01 , anova , post - test student t 
 or kruskal - wallis , post test student - newman - keuls ) ; c : groups 
 with n < 4 were not included in statistical analysis ; sd : standard 
 deviation . 
 

a : statistically significant in comparison to females of the 
 same age - class ( p = 0.01 , mann - whitney u test ) ; 
 b : statistically significant in relationship to controls 
 of the same age class ( p < 0.01 , anova , post - test student t 
 or kruskal - wallis , post test student - newman - keuls ) ; c : groups 
 with n < 4 were not included in statistical analysis ; sd : standard 
 deviation . 

the effect of cirrhosis on frequency of micronucleus ( mn ) , 
 nucleoplasmatic bridges ( npb ) and nuclear buds ( nbuds ) in males hepatitis b 
 virus ( hbv ) or hepatitis c virus ( hcv)-infected patientsgroupscirrhosis ( n)mn ( mean  sd)npb ( mean  sd)nbuds ( mean  sd ) 

 total patientsno ( 23)6.82  4.102.98  2.533.67  1.75yes ( 11)7.54  2.272.68  1.585.00  2.59hbvno ( 15)6.56  4.363.26  2.914.13  1.72yes ( 8)8.25  2.233.06  1.685.25  2.39hcvno ( 9)7.31  3.802.43  1.632.81  1.53yes ( 3)7.00  2.901.66 
cells from 15 chronically infected patients ( 9 hbv - infected and 6 hcv - infected patients ) 
 and 14 non - infected individuals were tested for dxr sensitivity .
the numbers of mn were 
 not significantly different between the hcv - infected and the hbv - infected patients ; 
 therefore , the patients were grouped together for comparison with the non - infected 
 individuals to improve the statistical analysis .
the patients exhibited significantly 
 higher numbers of mn in both untreated ( 6.76  3.03 , p = 0.03 ) and treated ( 15.03  
 4.94 , p = 0.001 ) cells compared with the cells of non - infected individuals ( 4.25  1.45 
 and 9.39  3.39 in untreated and treated cells , respectively ) .
dxr treatment 
 significantly increased the number of mn in both the control group ( p = 0.0002 ) and the 
 patient group ( p < 0.0001 ) .
the mean measurements of the induced dna damage were 
 higher in the patient group ( 8.30  5.40 ) than in the non - infected control group ( 5.18  
 3.45 ) . however , this difference was not found to be statistically significant , which was 
 possibly due to a type 2 error ( p = 0.06 ) .
chronic hepatotropic virus ( hbv and hcv ) infections are characterised by potentially 
 mutagenic cellular events , such as an increase in oxidative stress and viral integration 
 into the dna of the host cell ( farinati et al . 

, we demonstrated that the lymphocytes of hbv - infected patients and 
 hcv - infected patients exhibited an increased frequency of mn compared with lymphocytes 
 from healthy , non - infected individuals .
however , these authors reported a higher frequency of chromosome breaks , leading to the 
 formation of acentric chromosome / chromatid fragments that ultimately contributed to the 
 formation of mn .
furthermore , the mn may have resulted from an inability of whole 
 chromosomes to travel to the spindle poles during mitosis ( fenech et al .
. a higher frequency of chromosome gaps , 
 aneuploidy and polyploidy in the peripheral blood mononuclear cells ( pbmcs ) of 
 hcv - infected patients was reported previously ( machida 
 et al .
the present results suggest that chronic infection with hbv or 
 hcv accounts for chromosomal instability in lymphocytes and this phenomenon is 
 characterised by the formation of acentric fragments and/or aneuploidy .
npbs may 
 occur when dicentric chromosomes originating from chromosome breaks or 
 telomere - to - telomere end fusions are pulled to opposite poles of the cell during 
 mitosis .
nbuds are primarily considered to be formed from amplified dna that is being 
 eliminated from chromosomes ( fenech et al .
although gene amplifications ( myc and erbb2 ) were 
 demonstrated in hcc tissue samples that were infected with hbv or hcv ( al - qahtani et al .
2010 ) , no reports have described 
 this genetic alteration in pbmcs from individuals with viral hepatitis .
dicentric y 
 chromosomes have also been reported in the pbmcs of hcv - infected patients ( machida et al . 2010 ) .
together with our results , 
 these data suggest that the chromosomal instability in lymphocytes that results from hbv 
 infection ( and likely also from chronic hcv infection ) is characterised by the presence 
 of dicentric chromosomes and gene amplification . in this study ,
females and subjects aged  40 years exhibited more mn than did males and 
 subjects < 40 years , respectively , among both controls and hbv - infected patients . 
 although these differences were not statistically significant , the results are in 
 accordance with the well - known influence of sex and age on mn frequencies , as reported 
 before ( fenech 1998 , bonassi et al .
reports on the effects of sex and age on npbs and nbuds are 
 conflicting in the literature ( donmez - altuntas & 
 bitgen 2012 , nefic & handzic et al . 

, we found statistically significantly higher frequencies 
 of mn in males than in females ; however , females did not present cirrhosis in our 
 sample .
it is known that dna damage levels in the leukocytes of hbv - infected patients 
 and hcv - infected patients significantly correlate with the presence of liver lesions ( 
 farinati et al .
in fact , in 
 the current study , the frequencies of mn ( in hbv - infected patients ) and nbuds ( in 
 hbv - infected patients and hcv - infected patients ) were increased in cirrhotic males , but 
 this difference was not significant . whether the extension of liver lesions in male 
 patients is correlated with the frequency of mn found in lymphocytes is not completely 
 clear in the sample investigated here . even considering that the statistical analysis 
 may have been influenced by the small size of our sample ,
these results should be 
 considered with caution , especially because chromosome damage detected by the mn assay 
 is an important biomarker for cancer prediction ( bonassi 
 et al .
hbv and hcv may contribute to increased chromosomal aberrations in infected cells by 
 direct and indirect pathways .
regarding the indirect pathway , it has been shown that the 
 presence of reactive oxygen species ( ros ) resulting from cytokine activity during 
 chronic inflammation has a potent mutagenic effect ( yan 
 et al .
a relationship between infection with hbv or hcv and increased 
 production of ros , chromosomal aberrations and other dna damage has been reported 
 previously ( hagen et al . 1994 , machida et al . 2010 ) .
furthermore , leukocytes from 
 patients who are chronically infected with hbv or hcv exhibit higher levels of 8-ohdg , 
 which is the most frequent ros - induced base lesion ( farinati et al .
the 
 integration of hbv into the human genome affects the expression of genes located near 
 the site of insertion and also causes more widespread alterations of chromosomal 
 stability ( saigo et al .
2012 ) . because viral genome integration 
 into the host dna also frequently occurs in the pbmcs of
chronically hbv - infected 
 patients ( murakami et al . 2004 ) , this process 
 may contribute to genomic instability in these cells .
recently , it was demonstrated that 
 in chronic hcv infection , the presence of double strand breaks occurs concomitantly with 
 shortened telomeres in t lymphocytes .
this phenomenon is associated with the level of 
 fibrosis and may influence the response to treatment ( hoare et al . 2013 ) .
the frequency of mutations is directly influenced by the efficiency of the dna repair 
 mechanisms because failure to remove a lesion can facilitate mutational fixation . 

extensive evidence has demonstrated that proteins produced by hbv and hcv interact with 
 the proteins of the dna repair machinery and inhibit their functions in host cells ( 
 chen et al .
therefore , it is possible that the cells of infected patients exhibit 
 less efficient dna repair mechanisms due to the effects of the viral proteins , which may 
 contribute to the elevated frequency of dna damage detected in this study . 

au et al . ( 2010 ) reported that when the cells of 
 exposed populations ( in this case , hcv - infected patients or hbv - infected patients ) are 
 challenged with a dna - damaging agent in vitro , the in vivo exposure - induced repair 
 deficiency is dramatically amplified .
additionally , the deficiency will be detectable in 
 a challenge assay as an increase in the number of chromosomal aberrations , mn or 
 unrepaired dna strand breaks .
it has 
 been shown that b lymphocytes infected with hcv in vitro exhibit increased sensitivity 
 to bleomycin due to the action of the ns3 protein and the core viral protein ( machida et al .
furthermore , it was 
 demonstrated that lymphocytes from hbv or hcv - infected hcc patients exhibit an increased 
 sensitivity to bleomycin and benzo(a)pyrene - diol - epoxide , which is associated with an 
 increased risk of cancer development ( wu et al . 

the results obtained in the present study showed that the lymphocytes 
 of hbv - infected patients and hcv - infected patients are not more sensitive to dxr than 
 the lymphocytes of non - infected subjects .
however , a challenge assay with other 
 substances , such as bleomycin , needs to be performed to allow more definite conclusions 
 about mutagen sensitivity in these patients . in summary
, the present study demonstrated that the lymphocytes of patients who are 
 chronically infected with hbv or hcv exhibit greater chromosomal instability , 
 characterised by the presence of mn , npbs and nbuds .
although we did not observe a 
 statistically significant result , a possible influence of cirrhosis on these parameters 
 should be considered for further investigation .
 in this study , we analysed the frequency of micronuclei ( mn ) , nucleoplasmic bridges 
 ( npbs ) and nuclear buds ( nbuds ) and evaluated mutagen - induced sensitivity in the 
 lymphocytes of patients chronically infected with hepatitis b virus ( hbv ) or 
 hepatitis c virus ( hcv ) . in total , 49 patients with chronic viral hepatitis ( 28 
 hbv - infected and 21 hcv - infected patients ) and 33 healthy , non - infected blood donor 
 controls were investigated . 
 the frequencies (  ) of mn , npbs and nbuds in the controls 
 were 4.41  2.15 , 1.15  0.97 and 2.98  1.31 , respectively . 
 the frequencies of mn 
 and npbs were significantly increased ( p < 0.0001 ) in the patient group ( 7.01  
 3.23 and 2.76  2.08 , respectively ) compared with the control group . when considered 
 separately , the hbv - infected patients ( 7.18  3.57 ) and hcv - infected patients ( 3.27  
 2.40 ) each had greater numbers of mn than did the controls ( p < 0.0001 ) . the 
 hcv - infected patients displayed high numbers of npbs ( 2.09  1.33 ) and nbuds ( 4.38  
 3.28 ) , but only the hbv - infected patients exhibited a significant difference ( npbs = 
 3.27  2.40 , p < 0.0001 and nbuds = 4.71  2.79 , p = 0.03 ) in comparison with the 
 controls . 
 similar results were obtained for males , but not for females , when all 
 patients or the hbv - infected group was compared with the controls . 
 the lymphocytes of 
 the infected patients did not exhibit sensitivity to mutagen in comparison with the 
 lymphocytes of the controls ( p = 0.06 ) . 
 these results showed that the lymphocytes of 
 patients who were chronically infected with hbv or hcv presented greater chromosomal 
 instability . 