Ning Ma

Oxidative stress and its significant roles in neurodegenerative diseases and cancer.

Reactive oxygen and nitrogen species have been implicated in diverse pathophysiological conditions, including inflammation, neurodegenerative diseases and cancer. Accumulating evidence indicates that oxidative damage to biomolecules including lipids, proteins and DNA, contributes to these diseases. Previous studies suggest roles of lipid peroxidation and oxysterols in the development of neurodegenerative diseases and inflammation-related cancer. Our recent studies identifying and characterizing carbonylated proteins reveal oxidative damage to heat shock proteins in neurodegenerative disease models and inflammation-related cancer, suggesting dysfunction in their antioxidative properties. In neurodegenerative diseases, DNA damage may not only play a role in the induction of apoptosis, but also may inhibit cellular division via telomere shortening. Immunohistochemical analyses showed co-localization of oxidative/nitrative DNA lesions and stemness markers in the cells of inflammation-related cancers. Here, we review oxidative stress and its significant roles in neurodegenerative diseases and cancer.

Comparison of hepatic oxidative DNA damage in patients with chronic hepatitis B and C

Summary.u2002 8‐Hydroxydeoxyguanosine (8‐OHdG) is a promutagenic DNA lesion produced by hydroxyl radicals and is recognized as a useful marker in estimating DNA damage induced by oxidative stress. The aim of this study was to clarify the clinical significance of hepatic 8‐OHdG levels in patients with chronic viral hepatitis. Hepatic 8‐OHdG accumulation was investigated in patients with chronic hepatitis C (CH‐C) (nu2003=u200377) and chronic hepatitis B (CH‐B) (nu2003=u200334) by immunohistochemical staining of liver biopsy samples. 8‐OHdG positive hepatocytes were significantly higher in patients with CH‐C compared to CH‐B (median 55.0 vs 18.8 cells/105u2003μm2, Pu2003<u20030.0001). The number of positive hepatocytes significantly increased with the elevation of serum aminotransferase levels, especially in CH‐C patients (8‐OHdG vs alanine aminotransferase (ALT)/aspartate aminotrasferase (AST) were ru2003=u20030.738/0.720 in CH‐C and 0.506/0.515 in CH‐B). 8‐OHdG reactivity was strongly correlated with body and hepatic iron storage markers in CH‐C (vs serum ferritin, ru2003=u20030.615; vs hepatic total iron score, ru2003=u20030.520; vs hepatic hepcidin mRNA levels, ru2003=u20030.571), although it was related to serum HBV‐DNA titers (ru2003=u20030.540) and age of patients (ru2003=u2003–0.559) in CH‐B. These results indicate that hepatic oxidative DNA damage is common in chronic viral hepatitis, in particular chronic HCV‐infected patients, suggesting a possible link between chronic hepatic inflammation and hepatocarcinogenesis. The strong positive correlation between hepatic DNA damage and iron overload suggests that iron content is one of the most likely mediators of hepatic oxidative stress and iron reduction may be beneficial to reduce the incidence of hepatic cancer in CH‐C patients.

iNOS-dependent DNA damage via NF-κB expression in hamsters infected with Opisthorchis viverrini and its suppression by the antihelminthic drug praziquantel

Inflammation‐mediated DNA damage triggered by Opisthorchis viverrini (OV) infection is a major risk factor of cholangiocarcinoma (CCA). We have recently reported that nitrative and oxidative DNA damage participates in CCA development caused by repeated infection with OV [Pinlaor et al., Carcinogenesis 2004; 25:1535–42]. Therefore, to clarify the preventive effect of the antihelminthic drug praziquantel against cholangiocarcinogenesis, we assessed the effect of this drug on nitrative and oxidative DNA damage, including the formation of 8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG), and the expression of inducible nitric oxide synthase (iNOS) by immunohistochemistry in OV‐infected hamsters. We also examined the expression of nuclear factor‐κB (NF‐κB), which functions as a tumor promoter in inflammation‐associated cancer. Our results showed that although 1‐week treatment with praziquantel did not kill parasites completely in hamsters on days 14 and 30, this drug dramatically reduced inflammatory cell infiltration. Double immunofluorescence staining showed that drug treatment almost completely diminished OV‐induced 8‐nitroguanine and 8‐oxodG formation in bile duct epithelial cells. Quantitative analysis using an electrochemical detector coupled to HPLC revealed that 8‐oxodG level in the liver of OV‐infected hamsters was significantly decreased by drug treatment (p<0.05). Western blotting and immunohistochemistry revealed that the expression of NF‐κB and iNOS in bile duct epithelium was reduced by drug treatment. The amount of nitrate plus nitrite in the liver and plasma was significantly decreased after drug treatment. It is concluded that praziquantel can exhibit a preventive effect against OV‐induced cholangiocarcinoma by inhibiting iNOS‐dependent DNA damage through not only elimination of parasites but also a potential antiinflammatory effect.

Oxidative DNA Damage in Relation to Neurotoxicity in the Brain of Mice Exposed to Arsenic at Environmentally Relevant Levels

Oxidative DNA Damage in Relation to Neurotoxicity in the Brain of Mice Exposed to Arsenic at Environmentally Relevant Levels: Fengyuan Piao, et al. Department of Hygiene, Dalian Medical University, China—To clarify the association between oxidative DNA damage and the neurotoxicity of arsenic, the formation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) as an index of oxidative DNA damage in the brain was examined in mice fed with drinking water containing 1 or 2 ppm arsenic, using an HPLC‐electrochemical detector and immunohistochemical method. 8‐OHdG levels were significantly increased in the brain of mice given arsenic and its immunoreactivity was distributed in the cerebral and cerebellar cortexes. Cerebral cortex neurons and Purkinje cells in the cerebellar cortex showed degenerative changes in accordance with the distribution of 8‐OHdG immunoreactivity. The levels of arsenic in this study were lower than those reported in epidemiological studies. Thus, we conclude that environmentally relevant levels of arsenic induce pathological changes through oxidative DNA damage in the brain tissues in vivo and that cerebral and cerebellar cortex neurons seem to be the major targets of arsenic neurotoxicity.

Immunohistochemical evidences for localization and production of d-serine in some neurons in the rat brain

D-Amino acids are thought not to occur in mammalian tissues. However, previous studies reported D-serine was present only in astrocytes in the rat brain. In the present study, it was indicated by a highly sensitive immunocytochemical method with a D-serine specific antibody that D-serine was contained not only in astrocytes but also in some neurons, such as pyramidal neurons in the cerebral cortex, and neurons in the nucleus of the trapezoid body. Some amacrine cells also showed strong immunoreactivity for D-serine in the eyes which were injected with colchicine into the corpus vitreum.

Nitrative and oxidative DNA damage in cervical intraepithelial neoplasia associated with human papilloma virus infection

Recently, it was proposed that inflammation plays an integral role in the development of human papilloma virus (HPV)‐induced cervical cancer. The present study sought to examine if 8‐nitroguanine, a mutagenic nitrative DNA lesion formed during inflammation, contributes to cervical carcinogenesis. We obtained biopsy specimens from 30 patients with cervical intraepithelial neoplasia (CIN)1 (n = 9), CIN2 (n = 10), CIN3 (n = 6) and condyloma acuminatum (n = 5). We used immunohistochemistry to detect the formation of 8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG), an oxidative DNA lesion, and compared it with the expression of the cyclin‐dependent kinase inhibitor p16, which is considered to be a biomarker for cervical neoplasia. Double immunofluorescence labeling revealed that 8‐nitroguanine and 8‐oxodG were colocalized in cervical epithelial cells. Samples from CIN2–3 patients, most of whom were infected with high‐risk HPV subtypes, exhibited significantly more intense staining for 8‐nitroguanine than those with condyloma acuminatum. 8‐Nitroguanine and 8‐oxodG immunoreactivities correlated significantly with the CIN grade. We observed the expression of inducible nitric oxide synthase in epithelial and inflammatory cells from CIN lesions. Proliferating cell nuclear antigen was expressed specifically in dysplastic epithelial cells, but not in those of condyloma acuminatum. There were no statistically significant differences in p16 expression between CIN and condyloma acuminatum samples. These results suggest that high‐risk HPV types promote inducible nitric oxide synthase‐dependent DNA damage, which leads to dysplastic changes and carcinogenesis; in contrast, p16 appears to be merely a marker of HPV infection. Thus, 8‐nitroguanine is a more suitable and promising biomarker for evaluating the risk of inflammation‐mediated cervical carcinogenesis than p16. (Cancer Sci 2007; 98: 964–972)

Increased expression of multidrug resistance-associated protein 1 (mrp1) in hepatocyte basolateral membrane and renal tubular epithelia after bile duct ligation in rats

Components of the multidrug resistance-associated protein (mrp) family mediate the adenosine triphosphate (ATP)-dependent transport of conjugated organic anions in the liver. Of these, mrp1 and mrp2 have been shown to have similar substrate specificity and nucleotide sequence. The intracellular localization and distribution of mrp1 under normal condition and cholestasis have not been as yet completely elucidated. To clarify this point, in the present study we evaluated the intracellular localization of mrp1 in rat liver and kidney after bile duct ligation (BDL). Bile duct was ligated in Wistar rats. Sequential staining of mrp1 by immunofluorescence was carried out in rat liver and kidneys 1, 3, and 5 days after bile duct ligation using confocal laser scanning microscopy. Weak granular staining of mrp1 was observed in cytoplasm of control rat hepatocytes. In addition to increased cytoplasm staining of mrp1, belt-and granule-like staining of mrp1 in basolateral membrane of hepatocytes was also shown after BDL. Furthermore, mrp1 immunofluorescence increased over time after BDL. No specific immunoflurescence of mrp1 was detected in control rat kidney. However, mrp1-positive staining was observed in epithelia of some renal tubules after BDL. This study showed that mrp1 immunofluorescence increased in hepatocyte basolateral membrane and cytoplasm and epithelia of some renal tubules after BDL. This increased mrp1 expression may be an adaptive response to impairment of hepato-biliary organic anion transport during obstructive cholestasis.

Glial uptake of intracerebroventricularly injected d-serine in the rat brain: an immunocytochemical study

Recent studies have shown that free D-serine, an agonist for the N-methyl-D-aspartate receptor, is present in the forebrain of rats. In present study, we raised antibodies against D-serine and examined the distribution of both endogenous and intracerebroventricularly administered D-serine in the rat forebrain by an immunocytochemical method. D-Serine-like immunoreactive cells were found in glial cells in the brains of the rats injected with D-serine into the lateral ventricle. No immunoreactive cells were seen in the brains of untreated rats. The results suggest that glial cells may accumulate and catabolize D-serine to regulate the concentration of this neuroactive amino acid in the forebrain.

iNOS-dependent DNA damage in patients with malignant fibrous histiocytoma in relation to prognosis

Malignant fibrous histiocytoma (MFH) is one of the most common soft tissue sarcomas. MFH has been proposed to be a lesion accompanied with inflammatory responses. During chronic inflammation, reactive nitrogen and oxygen species generated from inflammatory cells are considered to participate in carcinogenesis by causing DNA damage. 8‐nitroguanine is a mutagenic nitrative DNA lesion formed during chronic inflammation. We examined whether nitrative DNA damage is related to the prognosis of MFH patients. We performed immunohistochemical analyses to examine the distribution of DNA damage and the expression of inflammation‐related molecules including inducible nitric oxide synthase (iNOS), nuclear factor‐κB (NF‐κB), and cyclooxygenase‐2 (COX‐2) in clinical specimens from 25 patients with MFH. We also analyzed the correlation of DNA damage or the expression of these genes with the prognosis of MFH patients. Immunohistochemical staining revealed that the formation of 8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG), an oxidative DNA lesion, occurred to a much greater extent in MFH tissue specimens from deceased patients than in live patients. iNOS, NF‐κB and COX‐2 were colocalized with 8‐nitroguanine in MFH tissues. It is noteworthy that the statistical analysis using the Kaplan‐Meier method demonstrated strong 8‐nitroguanine staining to be associated with a poor prognosis. In conclusion, 8‐nitroguanine appears to participate in not only the initiation and promotion of MFH, but also in the progression of MFH, and could therefore be used as a promising biomarker to evaluate the prognosis of cancer patients. (Cancer Sci 2007; 98: 163–168)

Reactive nitrogen species mediate DNA damage in Helicobacter pylori-infected gastric mucosa.

Background:u2002 Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can play an important role in cellular injury and carcinogenesis of gastric epithelial cells infected with Helicobacter pylori. 8‐OH‐deoxy guanosine (8‐OHdG) and 8‐nitroguanine (8‐NG) are markers for ROS‐ and RNS‐mediated DNA oxidation, respectively. In this study, RNS‐mediated DNA damage in gastric mucosa was observed directly using a newly developed antibody to 8‐NG to clarify how H. pylori infection causes nitrative DNA damage to gastric epithelial cells.