persistent hyperglycemia plays a major role in -cell dysfunction and insulin resistance , which leads to the further progression of diabetes . one potential central mechanism for hyperglycemia is the formation of excess reactive oxygen species ( ros ) . ros accumulation leads to oxidative dna damage , which is detected by several sensor proteins that activate dna repair pathways . imperfections in dna repair may induce changes in the genetic information , leading to cell death or disease , which implies that cell fate can be determined by the ability to repair dna . diabetes - associated dna damage has been widely described , and dna repair mechanisms are initiated to counteract hyperglycemia - induced oxidative dna damage . base excision repair ( ber ) is the major pathway involved in the repair of hyperglycemia - induced oxidative base modifications , which is different from the radiation - induced dna repair system , depending on a series of enzymatic reactions . several ber enzymes have been found to be related with diabetes . in the first step of the ber pathway , 8-oxoguanine dna glycosylase ( ogg1 ) recognizes and excises 8-hydroxylation of guanine bases ( 8-oxodg ) . the expression of ogg1 is significantly increased in the islets of patients with type 2 diabetes and positively correlates with disease progression . additionally , the activation of the dna repair enzyme , poly(adp - ribose ) polymerase 1 ( parp1 ) , is increased in diabetic patients compared to normal controls . while it is clear that hyperglycemia induces dna damage via the production of ros , the effect of hyperglycemia on dna repair enzymes has not been investigated . we therefore investigated whether the ber enzymes ogg1 , pol  , lig3 , xrcc1 , and parp1 were involved in glucose toxicity . as hyperglycemia - induced cellular dysfunction accumulates in vivo over a long period of time , the time courses of mrna expression of ber genes under high glucose treatment were investigated . the altered expression of ber mrnas in response to high glucose concentration may provide a model with which to further explore the molecular mechanisms of diabetes disease progression . human hepatoma hepg2 cells were obtained from the concord cell center ( peking union medical college , china ) and were cultured in eagle s minimum essential medium ( mem ; invitrogen , carlsbad , ca ) containing 5.5 mm glucose supplemented with 10% fetal bovine serum ( hyclone , logan , ut ) . cells were cultured in a 5% co2-humidified atmosphere at 37c . for high glucose treatment , ros determination was performed as previously described , based on the oxidation of the non - fluorescent dcfh ( beyotime , shanghai , china ) to the fluorescent dye 2,7-dichlorofluorescein ( dcf ) by peroxide . the intensity of fluorescence was immediately measured with flow cytometry ( bd , franklin lakes , nj ) at an excitation of 488 nm and emission of 530 nm . dna damage was analyzed by the alkaline single - cell gel electrophoresis comet assay , as previously described . the slides were observed under a microscope and the tail moment and olive tail moment were calculated from 100 randomly selected images from each sample using the casp comet assay software . total rna was extracted from hepg2 cells using trizol ( invitrogen ) , and cdna was synthesized from 2 g total rna using m - mulv reverse transcriptase ( new england biolabs , beverly , ma ) . real - time pcr was performed in a lightcycler ( applied biosystems , foster city , ca ) using the sybr green master mix kit . pcr primers were constructed for ogg1 , dna polymerase beta ( pol  ) , ligase iii ( lig3 ) , x - ray repair cross complementing group 1 ( xrcc1 ) and parp1 , based on the published nucleotide sequences of human gene regions . a list of the pcr primers is shown in table 1 . hprt served as an internal control , and the expression of the transcripts was quantified as a ratio of hprt expression . intracellular nad content was measured using the nad+/nadh quantification kit ( k337 ; biovision , mountain view , ca ) according to the manufacturer s instructions . samples ( 50 g protein ) were separated by 9% sds - page , transferred to pvdf membrane and blocked with 5% milk in tris - buffered saline with tween ( tbst ) . samples were then incubated overnight at 4c with antibodies for anti - poly(adp - ribose ) ( par ) ( trevigen , gaithersburg , md ) , anti - parp1 , anti - insulin receptor  ( insr  ) or anti - phospho - insr  ( p - tyr1150/1151-insr ) ( cell signaling technology , danvers , ma ) . bound antibodies were detected with hrp - linked secondary antibodies ( jackson immunoresearch laboratories , west grove , pa ) and visualized using enhanced chemiluminescence . to determine if insr was phosphorylated , we stimulated cells with 100 nm insulin for 20 min before lysis . comparisons between 2 means were performed using the independent - samples t - test ; multiple comparisons amongst groups were analyzed using one - way anova with spss software 11.0 . human hepatoma hepg2 cells were obtained from the concord cell center ( peking union medical college , china ) and were cultured in eagle s minimum essential medium ( mem ; invitrogen , carlsbad , ca ) containing 5.5 mm glucose supplemented with 10% fetal bovine serum ( hyclone , logan , ut ) . cells were cultured in a 5% co2-humidified atmosphere at 37c . for high glucose treatment , ros determination was performed as previously described , based on the oxidation of the non - fluorescent dcfh ( beyotime , shanghai , china ) to the fluorescent dye 2,7-dichlorofluorescein ( dcf ) by peroxide . the intensity of fluorescence was immediately measured with flow cytometry ( bd , franklin lakes , nj ) at an excitation of 488 nm and emission of 530 nm . dna damage was analyzed by the alkaline single - cell gel electrophoresis comet assay , as previously described . the slides were observed under a microscope and the tail moment and olive tail moment were calculated from 100 randomly selected images from each sample using the casp comet assay software . total rna was extracted from hepg2 cells using trizol ( invitrogen ) , and cdna was synthesized from 2 g total rna using m - mulv reverse transcriptase ( new england biolabs , beverly , ma ) . real - time pcr was performed in a lightcycler ( applied biosystems , foster city , ca ) using the sybr green master mix kit . pcr primers were constructed for ogg1 , dna polymerase beta ( pol  ) , ligase iii ( lig3 ) , x - ray repair cross complementing group 1 ( xrcc1 ) and parp1 , based on the published nucleotide sequences of human gene regions . hprt served as an internal control , and the expression of the transcripts was quantified as a ratio of hprt expression . intracellular nad content was measured using the nad+/nadh quantification kit ( k337 ; biovision , mountain view , ca ) according to the manufacturer s instructions . samples ( 50 g protein ) were separated by 9% sds - page , transferred to pvdf membrane and blocked with 5% milk in tris - buffered saline with tween ( tbst ) . samples were then incubated overnight at 4c with antibodies for anti - poly(adp - ribose ) ( par ) ( trevigen , gaithersburg , md ) , anti - parp1 , anti - insulin receptor  ( insr  ) or anti - phospho - insr  ( p - tyr1150/1151-insr ) ( cell signaling technology , danvers , ma ) . bound antibodies were detected with hrp - linked secondary antibodies ( jackson immunoresearch laboratories , west grove , pa ) and visualized using enhanced chemiluminescence . to determine if insr was phosphorylated , we stimulated cells with 100 nm insulin for 20 min before lysis . comparisons between 2 means were performed using the independent - samples t - test ; multiple comparisons amongst groups were analyzed using one - way anova with spss software 11.0 . no significant differences in ros accumulation were observed in cells cultured with 5.5 mm glucose for 1 , 2 or 4 days ( figure 1 ) or 24.5 mm - mannitol ( permeability control ) . extended exposure of hepg2 cells to 30 mm glucose induced ros accumulation and dna damage . compared to 5.5 mm glucose - treated cells ( control ) , 30 mm glucose led to a 3-fold increase in ros accumulation at day 4 ( figure 1 , p<0.05 ) . additionally , the extent of dna damage , indicated by the olive tail moment and tail moment , was increased in cells treated with 30 mm glucose compared to cells treated with 5.5 mm glucose ( figure 2 , p<0.05 ) . the mrna expression of ber proteins was examined in hepg2 cells cultured in 5.5 or 30 mm glucose for 1 to 4 days to assess how the dna repair pathway responded to dna damage during long - term high glucose treatment . the expression of the mrnas of ber genes did not significantly change in hepg2 cells cultured with 5.5 mm glucose at any time - point . in cells treated with high levels of glucose , the expression of ogg1 increased 1.8-fold at day 1 , which then decreased after day 2 , compared to cells cultured in 5.5 mm glucose ( p<0.05 , figure 3a ) . in cells treated with 30 mm glucose , the expression of pol  increased at day 1 ( 2.7-fold ) and day 2 ( 3.2-fold ) and decreased after day 4 , compared to cells treated with 5.5 mm glucose ( p<0.05 , figure 3b ) . lig3 mrna expression increased over time in 30 mm glucose - treated cells , with a 2-fold increase observed day 4 , compared to cells treated with 5.5 mm glucose ( p<0.05 , figure 3c ) . the mrna expression levels of both parp1 and xrcc1 were higher in cells treated with 30 mm glucose compared to cells treated with 5.5 mm glucose from day 1 to day 4 ; however , these differences were not significant ( figure 3d , e ) . since we have previously demonstrated that parp1 activation modulates insulin sensitivity through nad depletion , we examined the dynamic expression of parp1 activity , intracellular nad content and insulin receptor phosphorylation in high glucose concentrations . we determined the total poly(adp - ribosyl)ated protein content by western blotting as a measure of parp1 activity , and observed that parp1 activity in 30 mm glucose - treated cells was approximately 2.2-fold higher than in 5.5 mm glucose - treated cells after day 6 ( p<0.05 , figure 4b ) . additionally , 30 mm glucose reduced the intracellular nad content to approximately 50% of the nad content in 5.5 mm glucose - treated cells at day 4 ( p<0.05 , figure 4c ) . at day 2 , 30 mm glucose had no significant effect on cellular nad content and insulin receptor phosphorylation in response to insulin ( figure 4 ) . however , insulin receptor phosphorylation was markedly attenuated after 4 days of treatment with 30 mm glucose ( p<0.05 , figure 4d ) , which suggests that glucose toxicity accumulates gradually . no significant differences in ros accumulation were observed in cells cultured with 5.5 mm glucose for 1 , 2 or 4 days ( figure 1 ) or 24.5 mm - mannitol ( permeability control ) . extended exposure of hepg2 cells to 30 mm glucose induced ros accumulation and dna damage . compared to 5.5 mm glucose - treated cells ( control ) , 30 mm glucose led to a 3-fold increase in ros accumulation at day 4 ( figure 1 , p<0.05 ) . additionally , the extent of dna damage , indicated by the olive tail moment and tail moment , was increased in cells treated with 30 mm glucose compared to cells treated with 5.5 mm glucose ( figure 2 , p<0.05 ) . the mrna expression of ber proteins was examined in hepg2 cells cultured in 5.5 or 30 mm glucose for 1 to 4 days to assess how the dna repair pathway responded to dna damage during long - term high glucose treatment . the expression of the mrnas of ber genes did not significantly change in hepg2 cells cultured with 5.5 mm glucose at any time - point . in cells treated with high levels of glucose , the expression of ogg1 increased 1.8-fold at day 1 , which then decreased after day 2 , compared to cells cultured in 5.5 mm glucose ( p<0.05 , figure 3a ) . in cells treated with 30 mm glucose , the expression of pol  increased at day 1 ( 2.7-fold ) and day 2 ( 3.2-fold ) and decreased after day 4 , compared to cells treated with 5.5 mm glucose ( p<0.05 , figure 3b ) . lig3 mrna expression increased over time in 30 mm glucose - treated cells , with a 2-fold increase observed day 4 , compared to cells treated with 5.5 mm glucose ( p<0.05 , figure 3c ) . the mrna expression levels of both parp1 and xrcc1 were higher in cells treated with 30 mm glucose compared to cells treated with 5.5 mm glucose from day 1 to day 4 ; however , these differences were not significant ( figure 3d , e ) . since we have previously demonstrated that parp1 activation modulates insulin sensitivity through nad depletion , we examined the dynamic expression of parp1 activity , intracellular nad content and insulin receptor phosphorylation in high glucose concentrations . we determined the total poly(adp - ribosyl)ated protein content by western blotting as a measure of parp1 activity , and observed that parp1 activity in 30 mm glucose - treated cells was approximately 2.2-fold higher than in 5.5 mm glucose - treated cells after day 6 ( p<0.05 , figure 4b ) . additionally , 30 mm glucose reduced the intracellular nad content to approximately 50% of the nad content in 5.5 mm glucose - treated cells at day 4 ( p<0.05 , figure 4c ) . at day 2 , 30 mm glucose had no significant effect on cellular nad content and insulin receptor phosphorylation in response to insulin ( figure 4 ) . however , insulin receptor phosphorylation was markedly attenuated after 4 days of treatment with 30 mm glucose ( p<0.05 , figure 4d ) , which suggests that glucose toxicity accumulates gradually . the accumulation of oxidative damage as a result of chronic hyperglycemia is a causal link to tissue dysfunction in diabetes . dna oxidative damage can induce transcription and translation errors , which possibly contributed to pancreatic -cell dysfunction and insulin resistance . fortunately , the dna repair system repairs the damaged dna and maintains normal functions in cells . in this study we demonstrated that genes in the base excision repair pathway changed significantly in response to high glucose treatment , which contributed to altered dna repair ability and genome stability . ros generation is a basic event in oxidative damage , so in the first part of this study we demonstrated that glucose intake caused a significant increase in ros generation , which was consistent with a previous study by mohanty . however , in diabetes there are abnormalities of lipid metabolism and an increase in lipolysis , free fatty acid ( ffa ) concentrations and triglycerides . several studies have demonstrated that an increase in ffa concentrations induces ros generation , lipid peroxidation and inflammation . therefore , the oxidatively - mediated damage may be from a combination of hyperglycemia and elevated ffa concentrations . the source of ros generation is an exciting focus in diabetes research , and deserves further investigation . an increase in ogg 1 and pol  mrna levels was observed in hepg2 cells after 1-day culture in a high glucose environment ; this temporal increase may counteract dna damage induced by oxidative stress . this finding was consistent with the results of another report , in which pol  expression was induced by oxidizing agents . however , after long - term exposure to high glucose , both of them reduced to the level of the normal glucose group . our experiments also revealed that dna damage increased gradually with the time of high glucose treatment . high glucose treatment for 4 days caused the predominance of dna damage over dna repair defense system , leading to dna damage accumulation , which may induce genomic instability and cell dysfunction . the molecular mechanism of how high glucose modulates ber enzymes expression needs to be investigated further . previous studies have demonstrated that high glucose treatment decreases ogg1 expression via a redox - dependent activation of akt in murine proximal tubular epithelial cells , which provides a mechanism of oxidative stress - mediated dna damage in diabetes . simone et al .   also detected an initial increase in ogg1 expression , but the high glucose treatment time was short compared to our study , which may have been due to the use of different cell lines . we observed that the ber accessory protein mrnas , parp1 and xrcc1 , exhibited similar expression patterns in response to high glucose , but neither of them were significantly altered by high glucose treatment in this study . since parp1 activation has been proved to be related to diabetic complications , we then examined the expression and activity of parp1 . our results show that treatment with high levels of glucose increased parp1 activity , but did not significantly alter parp1 protein expression . this suggests that the modulation of parp1 expression by high glucose concentration is regulated in a post - translational manner . we have previously demonstrated that parp1 activation leads to the downregulation of intercellular nad content and insulin receptor ( insr ) phosphorylation in hepg2 cells . therefore , we also used intracellular nad content and insulin receptor phosphorylation level to evaluate the extent of high glucose damage . high glucose treatment decreased intercellular nad and insulin receptor phosphorylation in a time - dependent manner in a hepatoma cell line . these results confirm that long - term high glucose treatment gradually induces the accumulation of cellular dysfunction . the altered expression of ber protein mrnas in response to high glucose levels suggests functional roles for the ber repair system in genomic stability . in hyperglycemia , the accumulation of dna damage due to the imbalance between dna damage and repair may lead to further disease progression towards aggressive diabetes , which should be investigated in future studies . the altered expression of ber mrnas may have potential as biomarkers to estimate disease progression in patients with diabetes .
summarybackgroundit is widely accepted that chronic hyperglycemia induces dna oxidative damage in type 2 diabetes , but little is known about the effect of hyperglycemia on the dna repair system which plays a critical role in the maintenance of genomic dna stability in diabetes.material/methodsto investigate the alteration of base excision repair ( ber ) genes under hyperglycemia , the relative expression of the mrnas of the ber genes 
 ogg1 , pol , lig3 , xrcc1 , and parp1  were quantified using real - time pcr in hepg2 hepatocytes incubated with 5.5 mm or 30 mm glucose.resultshigh levels of glucose induced ros accumulation and dna damage , paralleling the dynamic alterations of ber mrna expression . 
 compared to 5.5 mm glucose - treated cells , ogg1 and pol mrna expression transiently increased at day 1 and decreased after day 4 in cells exposed to 30 mm glucose . 
 exposure to 30 mm glucose increased the activity of parp1 , which led to reduced cellular nad content and insulin receptor phosphorylation.conclusionsexposure to high concentrations of glucose initially led to the increased expression of ber mrnas to counteract hyperglycemia - induced dna damage ; however , long - term exposure to high glucose concentrations reduced the expression of mrna from ber genes , leading to accumulated dna damage .