oxidative stress is now recognized to be a prominent feature of many acute and chronic diseases and even of normal ageing processes [ 14 ] . dna and nucleotides are at high risk of being oxidized by reactive oxygen species ( ros ) , which are generated as byproducts of oxygen respiration or molecular executors in host defence , or through environmental exposure . among bases 8-hydroxy-2-deoxyguanosine ( 8-ohdg ) , one of the major dna base - modified products , may be induced by hydroxyl radicals , singlet oxygen , or photodynamic action . the formation of 8-ohdg in dna is frequently measured in serum , leukocytes , and urine to assess oxidative stress in humans [ 911 ] ; higher amounts have been detected in smokers and people exposed to environmental tobacco smoke compared with non - smokers [ 1214 ] . in metabolic syndrome , diabetes , and inflammatory diseases , oxidative stress and 8-ohdg level ( in blood and urine ) this compound is known to be mutagenic by mispairing with adenine ; thus , during the next round of replication , faultily paired adenine will pair with thymidine and transversion g : ct : a arises . increased oxidative stress chang et al . reported that the content of 8-ohdg in serum can act as a sensitive biomarker for colorectal carcinoma . its level was also found to be elevated in dna isolated from lymphocytes of colon carcinoma patients . sato et al . suggested that an increased plasma level of 8-ohdg is associated with development of colorectal adenoma and early cancer , but cancer tissues themselves are not always the major source . moreover , dincer et al . concluded that low plasma levels of 8-ohdg ( together with altered antioxidant activity ) may implicate the defective repair of oxidative dna damage in patients with colon and gastric cancer . in contrast , a study with more than 100 patients showed no significant differences at the individual level between the level of 8-ohdg in lymphocytes and the excretion of 8-ohdg measured by chromatographic methods . it is difficult to exclude all factors that could affect the status of oxidative dna damage and contribute to the increasing level of 8-ohdg in the entire human body . the direct measurement of 8-ohdg in the appropriate tissues can give a more precise picture of what is happening directly in the tissue . therefore , we decided to determine the 8-ohdg level directly in tumor and corresponding normal mucosa for comparison . the patients included in the study underwent surgery at the maria skodowska - curie , memorial cancer center and institute of oncology , gliwice branch , poland , during 20052007 . the study group consisted of 47 patients ( 27 men and 42 women ) diagnosed with sporadic colorectal adenocarcinoma . in this group the cancerous tissues and corresponding normal mucosa ( taken from at least 2 cm , mean 4.5 cm , distance from the tumor ) were resected . the age of the patients ranged from 26 to 82 years ( mean age : 6511 years ) . the coincidence of diabetes , lipid disorders , chronic alimentary tract diseases , and cigarette smoking were also exclusion criteria . twenty - eight patients ( 60% ) were diagnosed as a or b duke s stage and 19 ( 40% ) as c or d duke s stage . ethical approval for this study was received from the ethics committee of the center of oncology , maria skodowska - curie memorial institute of oncology in gliwice ( no . d0/dgp/493 - 10/05 ) and ( no . kb/493 - 54/07 ) . written informed consent the tumor and corresponding normal mucosa samples were frozen on dry ice and stored at 20c until needed for further investigation . the frozen tissue samples were homogenized with the fastprep-24 system ( mp biomedicals , usa ) using lysing matrix d ( mp biomedicals , usa ) . dna was isolated using the dneasy tissue kit ( qiagen , germany ) according to the provided protocol . then dna was hydrolyzed to nucleotides : dna samples were mixed with 40 mm sodium acetate ( poch , poland ) , 0.1 mm zncl2 ( merck , germany ) , ph 5.1 , and nuclease p1 ( sigma - aldrich , usa ) solution ( 20 g protein per sample ) . samples were incubated at 37c for 1 h. thereafter , 1 m tris - hcl buffer ( fluka , usa ) , ph=7.4 and alkaline phosphatase ( sigma - aldrich , usa ) solution containing 1.5 units of the enzyme were added to the samples for phosphate residue removal , and the samples were incubated for 1 h at 37c . all dna hydrolysates were ultrafiltered using amicon ultra-4 ( millipore , usa ) with a speed of 8000 rpm for 40 min at a temperature of 22c . isolated dna was prepared for high - performance liquid chromatography according to the procedure described by foksinski et al . , calculating the reagents volume for the amount of dna . high - performance liquid chromatography ( hplc ) with electrochemical ( ec ) and uv detectors was used for 8-ohdg and 2-dg ( 2-deoxyguanosine ) estimation . detection of dg was performed by uv detector ( dione , germany ) at 260 nm and 8-ohdg was determined by electrochemical detector ( gilson , france ) , for which an oxidation potential of + 700 mv was used ( an electrode set to sensitivity of 10 na / v ) . the mobile phase consisted of 50 mm kh2po4 ( poch , poland ) buffer ( ph 5.5 ) and methanol ( poch , poland ) . those components were well - proportioned in 9:1 ratio , based on the procedure elaborated by shigenaga et al . . the separations were performed at a flow rate of 0.7 ml / min , and the volume of loop equalled 20 l . 8-hydroxy-2-deoxyguanosine ( sigma - aldrich , usa ) and 2-deoxyguanosine ( fluka , usa ) were used as a standard for hplc . the amount of 8-ohdg in dna was calculated as the number of 8-ohdg molecules/10 unmodified 2-dg molecules . our data had a non - normal distribution , so we used a non - parametric test . for comparisons of 8-ohdg level in cancer tissue and corresponding mucosa samples , analyses of this parameter according to age , sex , and duke s staging were conducted with the mann - whitney u test . a p value of less than 0.05 was considered significant . the bonferroni correction for multiple comparisons was applied , and the corrected p value of less than 0.025 ( for 2 comparisons ) and less than 0.01 ( for 3 comparisons ) was considered statistically significant . the patients included in the study underwent surgery at the maria skodowska - curie , memorial cancer center and institute of oncology , gliwice branch , poland , during 20052007 . the study group consisted of 47 patients ( 27 men and 42 women ) diagnosed with sporadic colorectal adenocarcinoma . in this group the cancerous tissues and corresponding normal mucosa ( taken from at least 2 cm , mean 4.5 cm , distance from the tumor ) were resected . the age of the patients ranged from 26 to 82 years ( mean age : 6511 years ) . the coincidence of diabetes , lipid disorders , chronic alimentary tract diseases , and cigarette smoking were also exclusion criteria . twenty - eight patients ( 60% ) were diagnosed as a or b duke s stage and 19 ( 40% ) as c or d duke s stage . ethical approval for this study was received from the ethics committee of the center of oncology , maria skodowska - curie memorial institute of oncology in gliwice ( no . d0/dgp/493 - 10/05 ) and ( no . kb/493 - 54/07 ) . written informed consent the tumor and corresponding normal mucosa samples were frozen on dry ice and stored at 20c until needed for further investigation . the frozen tissue samples were homogenized with the fastprep-24 system ( mp biomedicals , usa ) using lysing matrix d ( mp biomedicals , usa ) . dna was isolated using the dneasy tissue kit ( qiagen , germany ) according to the provided protocol . then dna was hydrolyzed to nucleotides : dna samples were mixed with 40 mm sodium acetate ( poch , poland ) , 0.1 mm zncl2 ( merck , germany ) , ph 5.1 , and nuclease p1 ( sigma - aldrich , usa ) solution ( 20 g protein per sample ) . samples were incubated at 37c for 1 h. thereafter , 1 m tris - hcl buffer ( fluka , usa ) , ph=7.4 and alkaline phosphatase ( sigma - aldrich , usa ) solution containing 1.5 units of the enzyme were added to the samples for phosphate residue removal , and the samples were incubated for 1 h at 37c . all dna hydrolysates were ultrafiltered using amicon ultra-4 ( millipore , usa ) with a speed of 8000 rpm for 40 min at a temperature of 22c . isolated dna was prepared for high - performance liquid chromatography according to the procedure described by foksinski et al . , calculating the reagents volume for the amount of dna . high - performance liquid chromatography ( hplc ) with electrochemical ( ec ) and uv detectors was used for 8-ohdg and 2-dg ( 2-deoxyguanosine ) estimation . detection of dg was performed by uv detector ( dione , germany ) at 260 nm and 8-ohdg was determined by electrochemical detector ( gilson , france ) , for which an oxidation potential of + 700 mv was used ( an electrode set to sensitivity of 10 na / v ) . the mobile phase consisted of 50 mm kh2po4 ( poch , poland ) buffer ( ph 5.5 ) and methanol ( poch , poland ) . those components were well - proportioned in 9:1 ratio , based on the procedure elaborated by shigenaga et al . . the separations were performed at a flow rate of 0.7 ml / min , and the volume of loop equalled 20 l . 8-hydroxy-2-deoxyguanosine ( sigma - aldrich , usa ) and 2-deoxyguanosine ( fluka , usa ) were used as a standard for hplc . the amount of 8-ohdg in dna was calculated as the number of 8-ohdg molecules/10 unmodified 2-dg molecules . our data had a non - normal distribution , so we used a non - parametric test . for comparisons of 8-ohdg level in cancer tissue and corresponding mucosa samples , analyses of this parameter according to age , sex , and duke s staging were conducted with the mann - whitney u test . a p value of less than 0.05 was considered significant . the bonferroni correction for multiple comparisons was applied , and the corrected p value of less than 0.025 ( for 2 comparisons ) and less than 0.01 ( for 3 comparisons ) was considered statistically significant . the 8-ohdg level in the cancer tissues was significantly higher than in corresponding normal mucosa ( table 1 ) . more detailed analysis revealed that these differences were significant only in men and in patients over 65 years old . they were not documented for women and for patients under 65 years old ( table 1 ) . the differences between the 2 subgroups ( patients below 65 and over 65 years old ) were not significant within the normal mucosa ( p=0.77 ) or within the cancer tissues ( p=0.78 ) . analysis of 8-ohdg levels within normal mucosa and within cancer between women and men did not reveal significant differences ( p=0.283 and p=0.026 , respectively ) . a comparison between subgroups according to age and sex showed that 8-ohdg level was not significantly different in normal colonic mucosa or in cancer . patients with a and b colon adenocarcinoma stages were brought together , as well as c and d , and 2 subgroups ( a / b and c / d ) were analyzed between themselves in relation to 8-ohdg level . no statistical differences between normal mucosa and cancer tissue were confirmed in the a / b subgroup or in the c / d subgroup ( table 1 ) . the comparison of 8-ohdg level within cancer and corresponding normal mucosa in the cases of a / b and c / d stages showed no significant differences ( p=0.452 and p=0.871 , respectively ) . in recent studies , 8-ohdg has been widely used , not only as a biomarker indicating the level of endogenous oxidative dna damage , but also as a risk factor for many diseases , including cancer . a variety of analytical techniques used in assessing damage of oxidative dna exist . the high - performance liquid chromatography with electrochemical detection is one of the most widely used methods for quantitative analysis . in our study we used it for 8-ohdg estimation in tumor tissues and normal / adjacent mucosa in patients with colon adenocarcinoma . generally , we found that 8-ohdg levels were significantly higher in cancer tissue than in normal mucosa . park et al . reported high levels of 8-ohdg in colorectal tumour cells obtained by surgical resection . moreover , the imbalance of colon adenocarcinoma cancer tissue antioxidant potential compared with tissue without histopathologically detected neoplasmatic changes was previously described . our results also confirmed higher oxidative stress / inadequate activity of dna repair systems in colorectal cancer tissue , leading to an increase of local 8-ohdg concentrations . confusing results were obtained from data showing differences between 8-ohdg level in cancer tissue and normal mucosa in men and lack of significance in women and in patients over 65 years old , but not in younger patients . reported that androgen may increase oxidative stress ; however , kominea et al . found no difference in androgen receptor expression in gastric cancer of male and female subjects . another study confirmed that alternations in the androgen receptor signalling axis are central to castration - recurrent - prostate cancer . oxidative processes and the antioxidant enzymes activity , as well as efficiency of dna repair systems , are age - dependent parameters . using animal model experiment , provided indirect evidence that 8-ohdg might accumulate in a small pool of mitochondria , proportionally with increasing age . cancer is attributed to dna degeneration increasing exponentially with age and accumulating in the genome . it is assumed that 8-ohdg level in cancer tissue of older people reflects a cumulative effect of oxidative damage of dna as the result of both ageing and tumorigenesis . however , tsurudome et al . concluded that 8-ohdg level generally increase with age , and a similar effect is observed in colorectal cancer tissue , but without significant differences between patients with cancer and those with benign colorectal polyps . revealed higher 8-ohdg level in lymphocytes of patients with colorectal carcinoma compared with the healthy control group , but comparison by sex showed no differences . rainis et al . demonstrated an enhancement of oxidative stress in the neoplastic tissues of the colon , together with higher leukocyte activation within the carcinogenic tissue , suggesting a possible contribution of these cells to further oxidative stress - derived injury . data that should inform about oxidative stress in other tissues , in healthy men and women from 5 european countries ( france , ireland , the netherlands , spain , and the u.k . ) revealed a lower level in healthy women , stressing that mostly premenopausal women were examined . a comparison between subgroups according to age and sex in our study showed that 8-ohdg levels were no significantly different in normal colonic mucosa or in cancer . in both subgroups of patients , analysis of 8-ohdg level according to clinical staging estimated with duke s scale revealed the lack of differences between cancer and adjacent mucosa . no differences were found between a / b and c / d stages within normal colonic mucosa or within tumorous tissues . , who concluded that colorectal carcinoma cells are exposed to higher oxidative stress than corresponding non - tumorous epithelial cells , regardless of clinical stage . however , sato et al . showed that the plasma 8-ohdg level was not as high in patients with advanced cancer in comparison with patients with early cancer . we found that 8-ohdg levels were not related with age , sex , or tumor stage , possibly suggesting that higher 8-ohdg levels in colon adenocarcinoma tissue are not age , sex , or tumor stage dependent , but could be connected with a molecular mechanism within tumorous and corresponding normal mucosa . according to our best knowledge , similar results have not been reported so far , and some of our observations are difficult to explain . it was postulated that understanding the regulation of free - radical formation and its consequences regarding the oxidative repair phenotype and its relation to basic human differences like age and sex need further exploration . moreover , there are many factors ( dietary and non - dietary ) that influence dna oxidative damage and many of them could interfere with cancer tissue metabolism . and , last but not least , the effectiveness of several techniques measuring oxidative dna damage in various biological samples with likely spurious oxidation of dna during collection , assay , and/or storage of samples represents a real challenge . studies in the past 20 years have improved the quantitative estimation of 8-ohdg by various analytical techniques and have established it as a very important biomarker for carcinogenesis , aging , and degenerative diseases . sova et al . showed that low 8-oxodg level in serum and in breast cancer cells strongly indicate a more aggressive disease and concluded that negative 8-oxodg immunohistochemical staining is a powerful prognostic factor in breast carcinoma patients . in dna ( nuclear and mitochondrial ) , 8-ohdg is one of the predominant agents of free - radical - induced oxidative lesions . this is the reason why 8-ohdg has been used in many studies as a biomarker for the measurement of endogenous oxidative dna damage , and as a risk factor for many diseases such as cancer or degenerative diseases . the idea of using oxidative dna damage , like 8-ohdg , as a biomarker of oxidative stress , chronic inflammation , and susceptibility to cancer , gives a new perspective . there are many documented cases of higher level dna damage in malignant cells and tissues compared to non - malignant controls , but reactive oxygen species , produced either directly by tumors or indirectly via inflammatory responses , can cause dna damage in healthy neighboring cells as well as distant sites . therefore , 8-ohdg as a marker of oxidative dna damage and/or imbalance in antioxidant processes could be a useful parameter , but its role as a diagnostic and prognostic factor in colon adenocarcinoma seems to be limited . 8-ohdg reflects the local oxidative stress in colon adenocarcinoma tissue together with the ageing processes more than the intensity of tumorigenesis itself . because many factors could have an effect on oxidative modification of dna bases , its role as a diagnostic and/or prognostic factor in colon adenocarcinoma seems to be limited
background8-hydroxy-2-deoxyguanosine ( 8-ohdg ) is one of the most abundant oxidatively modified lesions in dna and is a marker of the oxidative stress . 
 8-ohdg is a mutagenic lesion and it can mispair with adenine , causing g : ct : a transversion . 
 our task was to determine the 8-ohdg level in patients with colorectal adenocarcinoma directly in tumor tissues and corresponding normal mucosa.material/methodssamples of tumor tissues and corresponding normal mucosa of 47 patients undergoing surgery for colorectal cancer were analyzed . 
 dna was isolated from both tumor and normal tissues . 
 then , dna was hydrolyzed to nucleotides using nuclease p1 and alkaline phosphatase . the 8-ohdg and 2-dg ( 2-deoxyguanosine ) 
 were determined in hydrolysates by high - performance liquid chromatography ( hplc ) with electrochemical ( ec ) and uv detector.resultsthe levels of 8-ohdg in colorectal adenocarcinoma tissues were higher than in corresponding normal mucosa . 
 no significant differences were shown in 8-ohdg levels in the cancerous and cancer - free tissues between age and sex and stages a / b and c / d of duke s classification.conclusions8-ohdg reflects the local oxidative stress in colon adenocarcinoma tissue together with ageing processes , but not the intensity of tumorigenesis itself . because of many factors that could influence the oxidative modification of dna bases , its role as a diagnostic and/or prognostic factor in colon adenocarcinoma seems to be limited .