chronic inflammation during gastroesophageal acid reflux disease ( gerd ) is an important risk factor of barrett 's esophagus ( be ) and esophageal carcinogenesis [ 1 , 2 ] . be is defined as the presence of a metaplastic columnar - lined esophagus induced by gerd . mutations of p53 are clearly involved in the pathogenesis of bea , and the fact that the mutations were detected in premalignant barrett 's epithelium supports the hypothesis that p53 mutations may be a useful marker for patients at increased risk for development of invasive cancer . reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) are generated during inflammation and considered to contribute to inflammation - mediated carcinogenesis [ 57 ] . ros and rns can induce the formation of 8-oxo-7,8-dihydro-2-deoxygaunosine ( 8-oxodg ) and 8-nitroguanine , the markers of oxidative and nitrative dna damage , respectively . production of nitric oxide ( no ) by inducible nitric oxide synthase ( inos ) and superoxide radical anion ( o2 ) by nad(p)h oxidase contributes to peroxynitrite ( onoo ) generation to cause 8-nitroguanine formation . abundant amount of no has been found in the human gastroesophageal junction , and no could diffuse into the adjacent epithelium at cytotoxic levels resulting in the pathogenesis of gerd spectrum . moreover , overexpression of inos and its transcriptional factor ( nfb ) was detected in the order of bea > be > normal esophagus tissues and the suppression of mn - sod expression was also found in be and bea tissues . these molecular events contribute to generation of onoo , resulting in the formation of dna lesions . recently , oxidative dna damage was also found to be associated with genetic instability via telomeric dysfunction , leading to p53 mutation and bea tumorigenesis . therefore , oxidative and nitrative stress has been shown to increase during the development from be to bea through the induction of ros- and rns - generating enzymes , leading to an increase in dna lesions , which contribute to mutations and genetic instability . accumulating evidence in recent years strongly indicates the existence of cancer stem cells in tumors of a wide variety of organs , particularly in inflammation - associated cancers . inflammation - associated tissue injury may activate stem ( progenitor ) cells , and multiple mutagenic and epigenetic changes are accumulated in these cells under such conditions . we have recently reported that oxidative and nitrative dna damage occurred in cells positive for stem cell markers in tissues of parasite - induced urinary bladder cancer   and intrahepatic cholangiocarcinoma , which are typical inflammation - related cancers . it is suggested that be is differentiated from adult stem cell lining at the basal layer of esophageal epithelium and bone marrow stem cells [ 1618 ] . cd133 is a transmembrane glycoprotein expressed in progenitor cells during differentiation and associated with cancer stem cells in several solid tumors [ 1923 ] . cd133 has the potential to differentiate benign tumors to malignant tumors in the tissues of barrett 's esophagus . these findings raise the possibility that cd133 could be used as a cancer stem cell marker related to oxidative and nitrative stress in bea . therefore , we examined the formation of inflammation - related dna lesions ( 8-nitroguanine and 8-oxodg ) and a stem cell marker ( cd133 ) in biopsy specimens of bea patients in comparison with those of normal esophagus and be tissues for understanding the mechanisms of gerd - induced esophageal carcinogenesis . all tissues used in this study were obtained from endoscopic biopsies or endoscopic mucosal resections from patients at tohoku university hospital as described previously . biopsy specimens were obtained from 19 be patients ( 14 males and 5 females , mean  sd , 63.6  12.4 years ) , 11 bea patients ( 10 males and 1 female , 66.5  12.8 years ) , and 7 subjects with normal esophagus ( 4 males and 3 females , 58.4  6.2 years ) . these specimens were formalin - fixed and embedded in paraffin . among bea patients , 10 cases were identified as stage i ( well - differentiated adenocarcinoma ) and 1 case was identified as stage ii ( moderately differentiated adenocarcinoma ) . in be patients , only those with histological confirmation of specialized intestinal metaplasia and three or more centimeters of macroscopic barrett 's epithelium were included . bea was defined by adenocarcinoma predominantly involving the tubular distal esophagus and histological evidence of adjacent barrett 's epithelium . in addition , subjects with macroscopically and histologically normal esophagus attending endoscopy for routine diagnostic procedure were recruited as controls . no participants administered acid suppression therapies , such as proton pump inhibitor or h2-blocker before endoscopic procedure . this study was approved by tohoku university hospital ethics committee ( number 2003 - 149 ) and written informed consent was obtained from all subjects . double or single fluorescent immunohistochemistry was performed to examine the colocalization of cd133 , 8-nitroguanine , and 8-oxodg as described previously . paraffin sections were incubated with the primary antibodies [ rabbit polyclonal anti - cd133 antibody ( 1  :  500 , abcam , cambridge , uk ) , rabbit polyclonal anti-8-nitroguanine antibody ( 1  g / ml ) produced by our group [ 25 , 26 ] , and mouse monoclonal anti-8-oxodg antibody ( 1  :  200 , japan institute for the control of aging , fukuroi , japan ) ] overnight at room temperature . the sections were next incubated with fluorescent secondary antibodies ( alexa 488-labeled goat anti - mouse igg and/or alexa 594-labeled goat anti - rabbit igg antibodies , 1  :  400 each , molecular probes inc . , eugene , oregon , usa ) for 3  h at room temperature . finally , the nuclei were stained by 4-6-diamidino-2-phenylindole ( dapi ) and the sections were examined with a fluorescence microscope ( lx70 , olympus , tokyo , japan ) or a laser scanning confocal microscope ( fluoview fv1000-d , olympus ) . we defined immunohistochemical grading ( ihc grading ) based on the intensity and frequency derived from the staining results in normal mucosal , columnar , and cancer cells of normal esophageal , be , and bea tissues , respectively , according to the method described in our previous reports [ 10 , 14 , 27 ] . the staining intensity was scored as negative ( 0 ) , weak ( + 1 ) , moderate ( + 2 ) , or strong ( + 3 ) . the frequency of positive cells in a section was scored as negative ( 0 ) , less than 25% ( + 1 ) , 2550% ( + 2 ) , 5175% ( + 3 ) , or more than 75% ( + 4 ) . ihc grading was assigned by an ihc score as follows :  , negative expression ( 0 ) ; + , weak expression ( 13 ) ; + + , moderate expression ( 46 ) ; + + + , high expression ( 79 ) ; or + + + + , very high expression ( 1012 ) . the ihc grading scores of each sample were approved by the expert pathologist . the statistically significant difference among normal , figure 1 shows the localization of cd133 in normal esophageal , be , and bea tissues examined by fluorescent immunohistochemistry . cd133 was not stained in normal esophageal tissues and its expression was significantly increased in be and bea tissues . interestingly , cd133 was weakly detected at apical surface of metaplastic columnar cells in be tissues and highly detected in cancer cells in bea tissues ( figure 1 , enlarged ; arrowheads ) . interestingly , cell membrane and cytoplasmic cd133 staining was detected only in cancer cells of bea tissues ( figure 1 , enlarged ; arrows ) . apical surface staining of cd133 was significantly higher in be and bea subjects compared with normal subjects ( table 1 , p = 0.005 and p = 0.027 , resp . ) , and there was significant difference between be and bea ( p = 0.038 ) . cd133 staining in the cytoplasm and cell membrane was observed in bea tissues alone and showed a significant increase compared with be tissues ( p = 0.001 ) . there was a nonsignificant difference in cd133 staining between normal and bea tissues ( p = 0.063 ) , probably because of small sample size . figure 2 shows the localization of 8-oxodg and 8-nitroguanine in normal esophageal , be , and bea tissues examined by fluorescent immunohistochemistry . 8-oxodg and 8-nitroguanine were weakly formed in nucleus of normal esophageal tissues , whereas they were highly formed in the nucleus of be and bea . cells positive for both 8-oxodg and 8-nitroguanine were significantly increased in be and bea tissues compared with normal subjects ( p < 0.001 and p = 0.001 , resp . ) , and tended to increase in bea subjects compared with be subjects ( p < 0.088 ) as shown in table 1 . our preliminary study indicated that phosphorylated h2ax ( -h2ax ) , as another dna damage marker , was observed in the nucleus of bea tissues ( supplementary figure  1 ) ( see supplementary material available online at http://dx.doi.org/10.1155/2016/7937814 ) . the oxidative dna damage marker ( 8-oxodg ) was found in cd133-positive cells in be and bea tissues as shown in figure 3 . moreover , the dna lesion was also formed in cell membrane and cytoplasmic cd133-positive cells . interestingly , the intensity of dna damage was significantly correlated with cd133 expression at the cytoplasm and cell membrane ( r = 0.405 , p = 0.013 by spearman rank correlation ) , whereas no correlation was found with cd133 expression at columnar apical surface . we showed here that dna lesions and the stem cell marker cd133 were colocalized in columnar gland cells in be tissues and cancer cells in bea tissues . 8-nitroguanine and 8-oxodg are mutagenic lesions leading to point mutation ( g to t transversion ) . these dna lesions could be detected in the nucleus of cancer stem - like cells in cholangiocarcinoma and bladder cancers , which may be involved in inflammation - driven carcinogenesis [ 14 , 15 , 27 ] . 8-oxodg and 8-nitroguanine were formed in the nucleus of several inflammation - related cancers such as liver fluke - associated cholangiocarcinoma . our previous studies confirmed the formation of 8-oxodg in the livers of liver fluke - infected hamster models , and the increase in cancer cells of human cholangiocarcinoma tissues   was detected by both immunohistochemistry and hplc coupled with electrochemical detector ( ecd ) . both techniques showed similar results , and therefore , we used immunohistochemical method in the present study , because of limited amounts of biopsy samples . the amount of 8-oxodg excreted in urine , which was measured by hplc - ecd , was significantly increased in parasite - infected subjects and cholangiocarcinoma patients compared with healthy control subjects . recently , elisa technique indicated that urinary 8-oxodg levels were correlated with hepatobiliary pathology of the liver fluke infection , which is associated with cholangiocarcinogenesis . accumulated evidence suggested that 8-oxodg and 8-nitroguanine could be used as the potential inflammation - related dna damage markers . our previous study reported that oxidative stress plays an important role in be and bea development . consequently , we have hypothesized that the formation of inflammation - related dna lesions in progenitor - like cells in be is involved in bea carcinogenesis . this process could be explained by the accumulation of mutation in progenitor cells , leading to the acquisition of the property of cancer stem cells . cd133 was originally identified as a transmembrane glycoprotein in normal hematopoietic stem and progenitor cells that participated in proliferation , self - renewal , and multilineage differentiation . cd133 has been characterized as a marker for tumor - initiating cells in gastrointestinal tract system including colon , gastric , pancreatic , and liver cancer [ 1519 , 26 ] . the present results indicated that cd133 expression was significantly increased in be and bea tissues compared with normal tissues , all of which were cd133-negative . cd133 was positive in cell membrane and the cytoplasm only in bea tissues but not in be tissues , and there was a significant difference between these groups as shown in table 1 . cd133 expression was observed in apical surface of columnar glands in be and bea tissues . our results are supported by a recent study showing that apical cd133 expression was observed in dysplastic barrett 's esophagus and esophageal adenocarcinoma . some other immunohistochemical studies showed the significance of cd133 expression in esophageal cancer [ 33 , 34 ] . the cytoplasmic localization of cd133 was also found in cholangiocarcinoma , which is also a cancer of epithelial cells . in addition , cd133 was detected in apical membrane of epithelial cells of normal minor salivary glands in normal subjects and adenoid cystic carcinoma patients . moreover , it was expressed in luminal ( apical ) surface membrane of gland - forming cells . cytoplasmic cd133 overexpression might be a useful marker of prognosis of gastric cancer [ 37 , 38 ] . sasaki et al . also reported that cytoplasmic expression of cd133 was a significant risk factor for the overall survival and tumor stages iii and iva of hepatocellular carcinoma patients . recently , nuclear and cytoplasmic cd133 was also detected in nonsmall cell lung cancer tissues and correlated with poor prognosis . from previous literatures and our results , it is speculated that cd133 expression in apical surface of epithelial cells means normal stem cell differentiation , whereas its expression in cell membrane and the cytoplasm is associated with the properties of cancer stem cells . therefore , cytoplasmic cd133 expression could be a marker of cancer stem cells in bea . the formation of mutagenic dna lesions , including 8-nitroguanine and 8-oxodg , was significantly and positively correlated with each cd133 staining pattern of cells in be and bea tissues . the proposed mechanism of be - derived esophageal carcinogenesis mediated by gerd is shown in figure 4 . gerd induces inflammatory responses and tissue injury , which mediate intestinal dysplasia and cd133 expression in apical surface of columnar epithelial cells . inflammatory responses also mediate dna damage in these cells with progenitor - like properties , which may lead to accumulation of mutations . under such conditions , the alteration in cd133 localization to cell membrane and the cytoplasm occurs , and the cells acquire the property of cancer stem cells , leading to bea development . this mechanism is supported by recent studies showing that chronic inflammation and following tissue damage may activate progenitor cells under ros- and rns - rich environment [ 14 , 15 , 27 , 41 ] . in conclusion , oxidative and nitrative dna lesions and differential cd133 localization would contribute to be - derived carcinogenesis , and these molecules could be used as potential biomarkers to evaluate the risk of this disease .
barrett 's esophagus ( be ) caused by gastroesophageal reflux is a major risk factor of barrett 's esophageal adenocarcinoma ( bea ) , an inflammation - related cancer . 
 chronic inflammation and following tissue damage may activate progenitor cells under reactive oxygen / nitrogen species - rich environment . 
 we previously reported the formation of oxidative / nitrative stress - mediated mutagenic dna lesions , 8-oxo-7,8-dihydro-2-deoxyguanosine ( 8-oxodg ) and 8-nitroguanine , in columnar epithelial cells of be tissues and cancer cells of bea tissues . 
 we investigated the mechanisms of bea development in relation to oxidative / nitrative dna damage and stem cell hypothesis . 
 we examined 8-nitroguanine and 8-oxodg formation and the expression of stem cell marker ( cd133 ) in biopsy specimens of patients with be and bea by immunohistochemical analysis in comparison with those of normal subjects . 
 cd133 was detected at apical surface of columnar epithelial cells of be and bea tissues , and the cytoplasm and cell membrane of cancer cells in bea tissues . 
 dna lesions and cd133 were colocalized in columnar epithelial cells and cancer cells . 
 their relative staining intensities in these tissues were significantly higher than those in normal subjects . 
 our results suggest that be columnar epithelial cells with cd133 expression in apical surface undergo inflammation - mediated dna damage , and mutated cells acquire the property of cancer stem cells with cytoplasmic cd133 expression .