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 .