digoxin is widely used in the treatment of various heart conditions , namely artial fibrillation , artial flutter and sometimes heart failure that can not be controlled by other medication ( chao et al . , 1992 ) .
several epidemiological studies indicated that breast cancer cells obtained from women on digitalis therapy ( for congestive heart failure ) were characterized by a series of more benign features compared with cancer cells from control patients ( stenkvist 1999 ; stenkvist et al .
the growth inhibitory effects of cardiac glycosides such as digoxin in the several cancer cell lines have been reported ( haux 1999 ; johansson et al . , 2001 ; lindholm et al . , 2002 ; lpez - lzaro et al . , 2005 ; svensson et al . , 2005 ; prassas and diamandis 2008 ) .
taken together , it is very probable that digoxin has effect(s ) on dna and chromosomes .   considering that there is no published data about effect of digoxin on induction of chromosomal aberrations , the present study was carried out .
the aim of the present study was to assess the effect of digoxin on chromatid and chromosomal aberrations on chinese hamster ovary ( cho ) cells .
in the present study , experiments were carried out using the chinese hamster ovary ( cho ) cell line .
the cells were maintained in rpmi-1640 medium ( from gibco ) supplemented with 10% inactivated fetal calf serum ( from gibco ) , 2 mm l - glutamine and with the addition of penicillin ( 100 u / ml ) and stereptomycin ( 100 mg / ml ) . for the present experiment we used lanoxin ( a trade name of digoxin , a liquid form , 0.25 mg / ml ; kern company ) .  
cells were seeded into 96-well plates at 2  10cells / well in 100 l complete medium .
after the cells clung to plates , the cells were treated with various concentration of digoxin ( 2.5 , 5 , 10 , 15 , 20 , 25 , 30 , 35 g / ml ) for 24 h. final volume in each well was 200 l .
subsequently , the cell viability was measured by mtt dye reduction assay ( mosmann , 1983 ; liu et al . , 1997 ) .
the formazan dye was measured by elisa reader . after obtaining the absorption , percentage inhibition of cell growth
the ic50 is defined as the cytotoxicity index that reduces the cell number to 50 % compared with untreated - control cho cells .  
mitomycin - c ( 0.06 g / ml ) and sodium arsenite ( 1 gm ) were used as positive controls ( mahata et al .
, 2004 ; celikler et al . , 2008 ; samuel et al . , 2011 ; sulaiman , 2012 ; sedigh - ardakani et al . , 2013 ) .
the cho cells were seeded at the density of 1.8  10 cells / petridish in the volume of 10 ml .
after 48 h , the cells were treated with different doses of digoxin ( 5 , 10 , 15 and 20 g / ml ) in rpmi-1640 for 24 h. chromosomes were conventionally stained with giemsa .  
chromosome aberrations were classified to chromatid breaks and polyploidy . to determine the mitotic index ( percentage of cells in mitosis ) , 1000 cells in each slide were observed . in each slide
all experiments ( for determining either cytotoxiciy or chromosomal breakage ) were performed in triplicate .
the level of the chromatid and chromosome breaks and also polyploidy , cells with aberrations and mitotic index are presented as mean + standard deviation ( sd ) .
comparisons of the mean values of the studied indices were done using one way analysis of variance .
statistical analysis was performed using spss statistical software package ( version 11.5 ) for windows ( spss inc . ,
in the present study , experiments were carried out using the chinese hamster ovary ( cho ) cell line .
the cells were maintained in rpmi-1640 medium ( from gibco ) supplemented with 10% inactivated fetal calf serum ( from gibco ) , 2 mm l - glutamine and with the addition of penicillin ( 100 u / ml ) and stereptomycin ( 100 mg / ml ) . for the present experiment we used lanoxin ( a trade name of digoxin , a liquid form , 0.25 mg / ml ; kern company ) .  
cells were seeded into 96-well plates at 2  10cells / well in 100 l complete medium .
after the cells clung to plates , the cells were treated with various concentration of digoxin ( 2.5 , 5 , 10 , 15 , 20 , 25 , 30 , 35 g / ml ) for 24 h. final volume in each well was 200 l .
subsequently , the cell viability was measured by mtt dye reduction assay ( mosmann , 1983 ; liu et al . , 1997 ) .
the formazan dye was measured by elisa reader . after obtaining the absorption , percentage inhibition of cell growth
the ic50 is defined as the cytotoxicity index that reduces the cell number to 50 % compared with untreated - control cho cells .  
mitomycin - c ( 0.06 g / ml ) and sodium arsenite ( 1 gm ) were used as positive controls ( mahata et al .
, 2004 ; celikler et al . , 2008 ; samuel et al . , 2011 ; sulaiman , 2012 ; sedigh - ardakani et al . , 2013 ) .
the cho cells were seeded at the density of 1.8  10 cells / petridish in the volume of 10 ml .
after 48 h , the cells were treated with different doses of digoxin ( 5 , 10 , 15 and 20 g / ml ) in rpmi-1640 for 24 h. chromosomes were conventionally stained with giemsa .  
chromosome aberrations were classified to chromatid breaks and polyploidy . to determine the mitotic index ( percentage of cells in mitosis ) , 1000 cells in each slide were observed . in each slide
all experiments ( for determining either cytotoxiciy or chromosomal breakage ) were performed in triplicate .
the level of the chromatid and chromosome breaks and also polyploidy , cells with aberrations and mitotic index are presented as mean + standard deviation ( sd ) .
comparisons of the mean values of the studied indices were done using one way analysis of variance .
statistical analysis was performed using spss statistical software package ( version 11.5 ) for windows ( spss inc . ,
effect of digoxin on proliferation of cho cells was investigated . after 24 h treatment of cho cells with 2.5 , 5 , 10 , 15 , 20 , 25 , 30 ,
35 g / ml of digoxin , the percent of cell inhibition were 4.5 , 9.4 , 19.4 , 28.3 , 39.9 , 60.8 , 83 , and 97 , respectively ( figure 1(fig .
1 ) ) . percent of growth inhibition rate increased as a function of digoxin concentration ( r = -0.980 , df = 25 , p < 0.001 ) .
the ic50 for digoxin was estimated 22.5  0.81 g / ml .   the results of chromosome aberration test were presented in table 1(tab . 1 ) .
there were significant difference between positive controls and negative control for frequencies of chromatid breaks and polyploidy ( for all comparisons p - values were less than 0.01 ) .
digoxin at final concentration of 25 g / ml results very low metaphases , therefore , the analysis did not perform .
all of the study aberrations and frequency of aberrant cells significantly increased as a function of digoxin concentration ( for chromatid breaks : r = 0.881 , df = 13 , p < 0.001 ; for polyploidy :
r = 0.777 , df = 13 , p = 0.001 ; for cells with aberrations : r = 0.926 , df = 13 , p < 0.001 ) .
however , the mitotic index negatively correlated with the concentration of digoxin ( r = -0.978 , df = 13 , p < 0.001 ) . based on
more than a thousand published scientific articles , it is well established that using in vitro system , cardiac glycosides showed anti - cancer properties ( prassas and diamandis , 2008 ) .
although the molecular mechanisms under lying the increased susceptibility to cancer cells to cardiac glycosides are not fully elucidated , some mechanisms such as alterations in homeostasis of k , na , and ca , inhibition of tumor necroses factor and nuclear factor b , increased production of reactive oxygen species , inhibition of toposiomerase ii , etc , might be involved ( mcconkey et al . , 2000 ; prassas and diamandis , 2008 ) .
considering that digoxin increased the frequency of chromatid and chromosome breaks , it is suggested that digoxin may interact with dna and subsequently inhibits cell division . the peak serum digoxin concentration ( 5 - 20 ng / ml ) was much lower than concentrations we used in the present experiments ( marcinkowska - krlewicz and feldmann , 1998 ; tayrouz et al . , 2003 ; kothare et al . , 2005 ) .
based on the results of the present study , all concentrations that cause chromosomal aberrations are in the cytotoxic range of digoxin .
therefore , it is possible that digoxin shows different patterns level of toxicity depended to cell type . on the other hand patients are exposed for longer periods compared to our cell culture system . taken together ,
it is necessary to evaluate the chromatid breaks , micronuclei , and sister chromatid exchange in lymphocytes of patients who received digoxin .  
finally it should be mentioned that digoxin must be used cautiously in generally and particularly in children and pregnant women .
 digoxin is a cardiac glycoside that has been reported to inhibit growth of multiple tumor cell types in vitro . 
 the present study was assessing the cytogenetic effects of this drug on chinese hamster ovary ( cho ) cells . 
 all experiments were performed in triplicate . 
 the ic50 was 22.5  0.8 g / ml . to investigate the clastogenic effects of drug , chromosomal aberration in metaphase cells 
 were analyzed . 
 chromatid breaks and polyploidy were the main types of aberration . 
 mitomycin - c and sodium arsenite were used as positive controls . 
 cho cells were exposed to different concentration of drug ( 5 , 10 , 15 , 20 g / ml ) in 24 hours . 
 all of the study aberrations and frequency of aberrant cells significantly increased as a function of digoxin concentration ( for chromatid breaks : r = 0.881 , df = 13 , p < 0.001 ; for polyploidy : 
 r = 0.777 , df = 13 , p = 0.001 ; for cells with aberrations : r = 0.926 , df = 13 , p < 0.001 ) . the mitotic index negatively correlated with the concentration of digoxin ( r = -0.978 , df = 13 , p < 0.001 ) . 
 all concentrations that cause chromosomal aberrations are in the cytotoxic range of the drug . the peak serum digoxin concentration ( 5 - 20 ng / ml ) was very lower than concentrations we used in the present experiments . 
 further studies on valuation of chromatid breaks , micronuclei , and sister chromatid exchange in lymphocytes of patients who received digoxin , were recommended . 
Introduction
Material and Methods
Cell culture and chromosome study
Statistical analysis
Result and Discussion
Acknowledgements
Disclosure Statement