comet lysis buffer ( 2.5 m sodium chloride , 100 mm edta ( ph 8.0 ) , 1% sarkosyl , 10 mm tris  hcl ( ph 8.0 ) , 10% dmso , 10% triton x-100 ) . comet alkali solution ( 0.3 m sodium hydroxide , 1 mm edta ) . cryopreservation medium ( 50% foetal calf serum [ fcs ] , 10% dmso , 40% dulbecco 's modified eagle 's medium [ dmem ] ) . culture medium for hepg2 cells ( dmem , 10% fcs , 50 u / ml penicillin , 50 u / ml streptomycin , 2 mm l - glutamine ) . all chemicals were purchased from sigma chemicals co. , dorset , uk . hepg2 cells were obtained from the european cell culture collection ( eccc , uk ) . hepg2 cells are human caucasian hepatocyte carcinoma cells taken from a primary hepatoblastoma ( liver biopsy ) from an 11-year - old male from argentina in 1979 . the frozen vials of the cells were held at room temperature for 1 min and then placed in a 37c incubator for 1 min until thawed and the cells were pipetted into a flask containing 10 ml of pre - warmed dmem . hepg2 cells were grown in dmem supplemented with 10% fcs , 2 mm glutamine , 100 g / ml penicillin and 100 g / ml streptomycin . the cells were fed three times a week and split when confluence was reached . to split the cells , the medium was removed , the cells were washed with hank 's buffered salt solution ( hbss ) , and then gently harvested with 1 ml detachment trypsin neutralising solution . once all the cells were loosened from the plate , 1 ml of detachment trypsin neutralising solution was added . the cell suspension was pipetted into a sterile falcon tube and centrifuged at 1,200 rpm for 5 min . the supernatant was removed and the pellet was gently resuspended in an appropriate volume of medium . cells were plated in a multi - well plate : 1 ml at 510 cells / ml in dmem . supplemented with 10% fcs , 2 mm glutamine , 100 g / ml penicillin and 100 g / ml streptomycin . the cells were allowed to attach for 24 h at 37c . then the medium was replaced by demem containing 25 m or 50 m h2o2 and duplicate cultures were incubated at 37c for 5 min , 30 min , 40 min , 1 h , or 24 h. at the end of incubation the cells were harvested and used in comet assay analysis of dna damage . the principle of the comet assay is that smaller dna molecules migrate faster in an electric field than larger molecules . the treated cells are encapsulated in gel and lysed by alkali , which also denatures the dna . subsequent electrophoresis causes migration of the dna . while the undamaged dna appears as a head , fragmented dna move faster , giving the characteristic appearance of a comet tail ( fig . 1 ) . levels of dna damage after exposure to hydrogen peroxide evaluated by the comet assay . 
 fig . partly frosted microscope slides were pre - coated with a smear of 1% low melting - point agarose ( lmpa ) and allowed to dry for 1 h at 37c . the slides were then placed on a bed of ice to pre - cool them before adding an agarose sandwich . an aliquot of 170 l of 1% lmpa was placed onto the slide and immediately covered with a coverslip . whilst this was setting , an aliquot of 170 l of the agarose / lymphocyte mix was placed on top of the first layer and again covered immediately with a coverslip . once set , the coverslip was removed and a final layer of 170 l 1% lmpa was applied to the existing gel to form an agarose sandwich , with the cells in the middle layer . the lights were switched off and the remainder of the assay was performed using indirect light . flowchart showing the comet assay for single - cell gel electrophoresis to determine dna damage ( 12 ) . a pyrex tray containing 500 ml of ice - cold lysis buffer was placed onto a bed of ice and the slides were gently lowered into it . after 1 h , the slides were removed and washed by placing them in 500 ml ice - cold pbs ( again in a pyrex tray on a bed of ice ) and allowing them to sit for 15 min . this removed the salt from the lysis buffer and prepared the slides for the next step . a horizontal electrophoresis tank was surrounded by ice and filled with 2 l of ice - cold lysis buffer . then the slides were gently lowered into a horizontal electrophoresis tank filled with 2 l of cold lysis buffer and surrounded by ice . the slides were placed for 30 min to allow the dna to unwind in the alkali buffer . next , the slides were removed and rinsed by placing them in 500 ml of ice - cold neutralising buffer for 10 min and then in 500 ml of ice - cold pbs for 15 min . dna damage was measured using the comet assay and expressed as olive tail moment ( otm ) . results are given as meansem for the 50 cells ( 25 per slide ) . to determine cell viability ( 14 ) , 100 l of cell suspension was mixed 1:1 with trypan blue stain ( 0.4% w / v , biowhittaker ) and pipetted into a neubauer haemocytometer ( vwr scientifics , west chester , pa ) . if membrane integrity has been compromised ( dead cells ) , the cells absorb the dye and appear blue . in our study , if cell viability < 80% , the cells were discarded and a new batch was started . for the experiments we report , one - way anova was performed when more than two groups were compared with a single control . differences between individual groups were assessed by a dunnett post hock test , using prism software ( version 4 ) . the slide was visualised using either a biorad mrc 600 confocal microscope or a leica tcs sp2 uv confocal microscope . images were obtained with the biorad using a krypton / argon laser , 20 magnification , and excitation wavelength at 568 nm , lens aperture of 0.4 and z series slices of 3-m steps . images were obtained with the leica using a 543 helium / neon ( he / ne ) laser at 55% power and 20 magnification , and the emission was collected using a detection window of 570655 nm wavelength . comet lysis buffer ( 2.5 m sodium chloride , 100 mm edta ( ph 8.0 ) , 1% sarkosyl , 10 mm tris  hcl ( ph 8.0 ) , 10% dmso , 10% triton x-100 ) . comet alkali solution ( 0.3 m sodium hydroxide , 1 mm edta ) . cryopreservation medium ( 50% foetal calf serum [ fcs ] , 10% dmso , 40% dulbecco 's modified eagle 's medium [ dmem ] ) . culture medium for hepg2 cells ( dmem , 10% fcs , 50 u / ml penicillin , 50 u / ml streptomycin , 2 mm l - glutamine ) . all chemicals were purchased from sigma chemicals co. , dorset , uk . hepg2 cells were obtained from the european cell culture collection ( eccc , uk ) . hepg2 cells are human caucasian hepatocyte carcinoma cells taken from a primary hepatoblastoma ( liver biopsy ) from an 11-year - old male from argentina in 1979 . the frozen vials of the cells were held at room temperature for 1 min and then placed in a 37c incubator for 1 min until thawed and the cells were pipetted into a flask containing 10 ml of pre - warmed dmem . hepg2 cells were grown in dmem supplemented with 10% fcs , 2 mm glutamine , 100 g / ml penicillin and 100 g / ml streptomycin . the cells were fed three times a week and split when confluence was reached . to split the cells , the medium was removed , the cells were washed with hank 's buffered salt solution ( hbss ) , and then gently harvested with 1 ml detachment trypsin neutralising solution . once all the cells were loosened from the plate , 1 ml of detachment trypsin neutralising solution was added . the cell suspension was pipetted into a sterile falcon tube and centrifuged at 1,200 rpm for 5 min . the supernatant was removed and the pellet was gently resuspended in an appropriate volume of medium . cells were plated in a multi - well plate : 1 ml at 510 cells / ml in dmem . supplemented with 10% fcs , 2 mm glutamine , 100 g / ml penicillin and 100 g / ml streptomycin . the cells were allowed to attach for 24 h at 37c . then the medium was replaced by demem containing 25 m or 50 m h2o2 and duplicate cultures were incubated at 37c for 5 min , 30 min , 40 min , 1 h , or 24 h. at the end of incubation the cells were harvested and used in comet assay analysis of dna damage . the principle of the comet assay is that smaller dna molecules migrate faster in an electric field than larger molecules . the treated cells are encapsulated in gel and lysed by alkali , which also denatures the dna . subsequent electrophoresis causes migration of the dna . while the undamaged dna appears as a head , fragmented dna move faster , giving the characteristic appearance of a comet tail ( fig . 1 ) . levels of dna damage after exposure to hydrogen peroxide evaluated by the comet assay . 
 fig . partly frosted microscope slides were pre - coated with a smear of 1% low melting - point agarose ( lmpa ) and allowed to dry for 1 h at 37c . the slides were then placed on a bed of ice to pre - cool them before adding an agarose sandwich . an aliquot of 170 l of 1% lmpa was placed onto the slide and immediately covered with a coverslip . whilst this was setting , an aliquot of 170 l of the agarose / lymphocyte mix was placed on top of the first layer and again covered immediately with a coverslip . once set , the coverslip was removed and a final layer of 170 l 1% lmpa was applied to the existing gel to form an agarose sandwich , with the cells in the middle layer . the lights were switched off and the remainder of the assay was performed using indirect light . flowchart showing the comet assay for single - cell gel electrophoresis to determine dna damage ( 12 ) . a pyrex tray containing 500 ml of ice - cold lysis buffer was placed onto a bed of ice and the slides were gently lowered into it . after 1 h , the slides were removed and washed by placing them in 500 ml ice - cold pbs ( again in a pyrex tray on a bed of ice ) and allowing them to sit for 15 min . this removed the salt from the lysis buffer and prepared the slides for the next step . a horizontal electrophoresis tank was surrounded by ice and filled with 2 l of ice - cold lysis buffer . then the slides were gently lowered into a horizontal electrophoresis tank filled with 2 l of cold lysis buffer and surrounded by ice . the slides were placed for 30 min to allow the dna to unwind in the alkali buffer . next , the slides were removed and rinsed by placing them in 500 ml of ice - cold neutralising buffer for 10 min and then in 500 ml of ice - cold pbs for 15 min . dna damage was measured using the comet assay and expressed as olive tail moment ( otm ) . to determine cell viability ( 14 ) , 100 l of cell suspension was mixed 1:1 with trypan blue stain ( 0.4% w / v , biowhittaker ) and pipetted into a neubauer haemocytometer ( vwr scientifics , west chester , pa ) . if membrane integrity has been compromised ( dead cells ) , the cells absorb the dye and appear blue . in our study , if cell viability < 80% , the cells were discarded and a new batch was started . for the experiments we report , cell viability was 9095% after culturing and before starting the comet assay . one - way anova was performed when more than two groups were compared with a single control . differences between individual groups were assessed by a dunnett post hock test , using prism software ( version 4 ) . the slide was visualised using either a biorad mrc 600 confocal microscope or a leica tcs sp2 uv confocal microscope . images were obtained with the biorad using a krypton / argon laser , 20 magnification , and excitation wavelength at 568 nm , lens aperture of 0.4 and z series slices of 3-m steps . images were obtained with the leica using a 543 helium / neon ( he / ne ) laser at 55% power and 20 magnification , and the emission was collected using a detection window of 570655 nm wavelength . cell viability following different treatments was 9095% and no floating cells were noticed in the medium . we analysed dna damage by the comet assay . following single - cell electrophoresis , the lengths of the comets ( dna trails ) depended on the treatment , with longer tails indicating more dna damage . however , cells incubated with h2o2 for 24 h appeared similar to untreated control cells ( fig . comet assay of the effect of duration of incubation with hydrogen peroxide on dna damage : untreated hepg2 cells ( a ) and hepg2 cells treated with 50 m h2o2 for 5 min ( b ) , 1 h ( c ) or 24 h ( d ) . the extent of dna damage , measured in otm , increased rapidly and significantly ( p<0.001 ) from baseline levels of 1.4 otm0.2 sem to 13.41.3 otm after 5 min of treatment with 25 m h2o2 and 15.50.6 otm after 5 min of treatment with 25 m h2o2 ( fig . the extent of damage increased further with time but at a slower rate , reaching about 2530 otm after 1 h of treatment . however , at the 24-h time - point the values for treatment with 25 and 50 m h2o2 ( 5.91.3 and 3.70.6 otm , respectively ) were not significantly higher than the baseline levels . the extent of dna damage was higher for cells treated with 50 m h2o2 than for those treated with a 25 m concentration at the 5 , 30 , 40 and 60 min time - points ( fig . hepg2 cells were treated with 25 m or 50 m h2o2 for 5 , 30 , 40 min , 1 h or 24 h and dna damage was assessed by the comet assay . the values are the meansem of 50 cells from two pooled wells ( 25 cells per slide ) . when formation of ros exceeds the biological defence capacity , the result is oxidative stress and tissue injury ( 15 ) . h2o2 can cause oxidative stress because it uses water channels ( aquaporins ) to rapidly cross - cell membranes ( 20 ) , reach the nucleus and cause damage to dna by generating hydroxyl - free radicals ( oh ) ( 21 ) . these radicals attack dna at the sugar residue of the dna backbone , leading to single strand breaks . they also modify purines and pyrimidines to their hydroxyl derivatives , such as 8-hydroxyguanine ( 16 ) . one study examined dna damage resulting from treatment of cultured human leukocytes with h2o2 concentrations of 25 , 50 , 100 and 200 m for 4 h. dna damage , assessed by the comet assay , increased significantly with increasing dose ( 17 ) . in our study , we treated hepg2 cells with 25 m or 50 m h2o2 for different durations : 5 min , 30 min , 40 min or 1 h. dna damage was both dose and duration dependent . dna damage increased with increasing time of incubation with h2o2 , but only between 5 and 60 mins of incubation . after 24 h of incubation with h2o2 , the extent of dna damage was not different from that in control cells , quite likely due to the action of dna repair mechanisms . as we did not use incubation times longer than an hour but shorter than 24 h , we do not know at what time point the effects of such repair mechanisms became evident . however , a similar study on colonocytes ( 18 ) showed that at a h2o2 concentration of 15 mm the dna damage was reduced when incubation times were > 30 min . similarly , treatment of human lymphocytes with 20 m h2o2 for 5 min induced dna damage but incubation of the cells for 2 h led to a considerable decrease in dna damage ( 19 ) . based on these reports we suggest that dna damage in our setup starts to decrease shortly after 1 h of incubation . in conclusion , the extent of dna damage was dose - dependent and incubation time - dependent . dna damage increased with time but later decreased , likely due to metabolism of h2o2 to water and the effects of dna repair . the authors have not received any funding or benefits from industry to conduct this study .