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Indium compounds have been widely used in manufacturing displays of mobile phones, computers and televisions. However, inhalation exposure to indium compounds causes interstitial pneumonia in exposed workers and lung cancer in experimental animals. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed under inflammatory conditions and may participate in indium-induced carcinogenesis. In this study, we examined 8-nitroG formation in A549 cultured human lung epithelial cells treated with indium compounds, including nanoparticles of indium oxide (In2O3) and indium-tin oxide (ITO), and indium chloride (InCl3). We performed fluorescent immunocytochemistry to examine 8-nitroG formation in indium-exposed A549 cells. All indium compounds significantly increased 8-nitroG formation in A549 cells at 5 ng/ml after 4 h incubation. 8-NitroG formation was largely reduced by 1400 W, methyl-β-cyclodextrin (MBCD) and monodansylcadaverine (MDC), suggesting the involvement of nitric oxide synthase and endocytosis. 8-NitroG formation in A549 cells was also largely suppressed by small interfering RNA (siRNA) for high-mobility group box-1 (HMGB1), receptor for advanced glycation and end products (AGER, RAGE) and Toll-like receptor 9 (TLR9). These results suggest that indium compounds induce inflammation-mediated DNA damage in lung epithelial cells via the HMGB1-RAGE-TLR9 pathway. This mechanism may contribute to indium-induced genotoxicity in the respiratory system.

Acrylamide is formed during the heating of food and is also found in cigarette smoke. It is classified by the International Agency for Research on Cancer as a probable human carcinogen (Group 2A). Glycidamide, an epoxide metabolite of acrylamide, is implicated in the mechanism of acrylamide carcinogenicity. Acrylamide causes oxidative DNA damage in target organs. We sought to clarify the mechanism of acrylamide-induced oxidative DNA damage by investigating site-specific DNA damage and reactive oxygen species (ROS) generation by a putative metabolite of acrylamide, acrylohydroxamic acid (AA). Our results, using 32P-5’-end-labeled DNA fragments, indicated that, although AA alone did not damage DNA, AA treated with amidase induced DNA damage in the presence of Cu(II). DNA cleavage occurred preferentially at T and C, and particularly at T in 5’-TG-3’ sequences, and the DNA cleavage pattern was similar to that of hydroxylamine. The DNA damage was inhibited by methional, catalase, and Cu(I)-chelator bathocuproine, suggesting that H2O2 and Cu(I) are involved in the mechanism of DNA damage induced by AA treated with amidase. In addition, amidase-treated AA increased 8-oxo-7,8-dihydro-2’-deoxyguanosine formation in calf thymus DNA, an indicator of oxidative DNA damage, in a dose-dependent manner. In conclusion, hydroxylamine, possibly produced from AA treated with amidase, was autoxidized via the Cu(II)/Cu(I) redox cycle and H2O2 generation, suggesting that oxidative DNA damage induced by ROS plays an important role in acrylamide-related carcinogenesis.

Naphthalene is a carcinogenic polycyclic aromatic hydrocarbon, to which humans are exposed as an air pollutant. Naphthalene is metabolized in humans to reactive intermediates such as 1,2-hydroxynaphthalene (1,2-NQH2), 1,4-NQH2, 1,2-naphthoquinone (1,2-NQ), and 1,4-NQ. We examined oxidative DNA damage by these naphthalene metabolites using 32P-labeled DNA fragments from human cancer-relevant genes. 1,2-NQH2 and 1,4-NQH2 induced DNA damage in the presence of Cu(II). The DNA-damaging activity of 1,2-NQH2 was significantly increased in the presence of the reduced form of nicotinamide adenine dinucleotide (NADH), whereas that of 1,4-NQH2 was not. In the presence of NADH, 1,2-NQ induced Cu(II)-dependent DNA damage, whereas 1,4-NQ did not. The calculated energy of the lowest unoccupied molecular orbital (LUMO), which corresponds to the reduction potential, was estimated to be −0.67 eV for 1,2-NQ and −0.75 eV for 1,4-NQ. These results suggest that 1,2-NQ was reduced more easily than 1,4-NQ. Furthermore, 1,2-NQH2, 1,4-NQH2, and 1,2-NQ plus NADH formed 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) as an oxidative DNA marker. Catalase and bathocuproine inhibited DNA damage, suggesting that H2O2 and Cu(I) were involved. These results indicate that NQH2s are oxidized to the corresponding NQs via semiquinone radicals, and that H2O2 and Cu(I) are generated during oxidation. 1,2-NQ is reduced by NADH to form the redox cycle, resulting in enhanced DNA damage. The formation of the corresponding semiquinone radicals was supported by an electron paramagnetic resonance (EPR) study. In conclusion, the redox cycle of 1,2-NQ/1,2-NQH2 may play a more important role in the carcinogenicity of naphthalene than that of 1,4-NQ/1,4-NQH2.

Morin is a potential inhibitor of amyloid β-peptide aggregation. This aggregation is involved in the pathogenesis of Alzheimer's disease. Meanwhile, morin has been found to be mutagenic and exhibits peroxidation of membrane lipids concurrent with DNA strand breaks in the presence of metal ions. To clarify a molecular mechanism of morin-induced DNA damage, we examined the DNA damage and its site specificity on 32P-5'-end-labeled human DNA fragments treated with morin plus Cu(II). The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, was also determined in calf thymus DNA treated with morin plus Cu(II). Morin-induced DNA strand breaks and base modification in the presence of Cu(II) were dose dependent. Morin plus Cu(II) caused piperidine-labile lesions preferentially at thymine and guanine residues. The DNA damage was inhibited by methional, catalase and Cu(I)-chelator bathocuproine. The typical •OH scavengers ethanol, mannitol and sodium formate showed no inhibitory effect on DNA damage induced by morin plus Cu(II). When superoxide dismutase was added to the solution, DNA damage was not inhibited. In addition, morin plus Cu(II) increased 8-oxodG formation in calf thymus DNA fragments. We conclude that morin undergoes autoxidation in the presence of Cu(II) via a Cu(I)/Cu(II) redox cycle and H2O2 generation to produce Cu(I)-hydroperoxide, which causes oxidative DNA damage.

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.

Infection and chronic inflammation have been recognized as important factors for carcinogenesis. Under inflammatory conditions, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated from inflammatory and epithelial cells, and result in the formation of oxidative and nitrative DNA lesions, such as 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and 8-nitroguanine. The DNA damage can cause mutations and has been implicated in inflammation-mediated carcinogenesis. It has been estimated that various infectious agents are carcinogenic to humans (IARC group 1), including bacterium Helicobacter pylori (H. pylori), viruses [hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV) and Epstein-Barr virus (EBV)] and parasites [Schistosoma haematobium (SH) and Opisthorchis viverrini (OV)]. H. pylori, HBV/HCV, HPV, EBV, SH and OV are important risk factors for gastric cancer, hepatocellular carcinoma, nasopharyngeal carcinoma, bladder cancer, and cholangiocarcinoma, respectively. We demonstrated that 8-nitroguanine was strongly formed via inducible nitric oxide synthase (iNOS) expression at these cancer sites of patients. Moreover, 8-nitroguanine was formed in Oct3/4-positive stem cells in SH-associated bladder cancer tissues, and in Oct3/4- and CD133-positive stem cells in OV-associated cholangiocarcinoma tissues. Therefore, it is considered that nitrative and oxidative DNA damage in stem cells may play a key role in infection-related carcinogenesis via chronic inflammation.

Nitrative and oxidative DNA damage plays an important role in inflammation-related carcinogenesis. Chronic inflammation such as parasite infection and primary sclerosing cholangitis can be an etiological factor of cholangiocarcinoma. Using a proteomic approach and double-fluorescent staining, we identified high expression and colocalization of albumin and cytokeratin-19 in liver fluke-associated cholangiocarcinoma tissues, compared with normal livers from cholangiocarcinoma patients and cadaveric donors, respectively. Albumin was detected not only in cells of hyperplastic bile ducts and cholangiocarcinoma, but also in liver stem/progenitor cell origin, such as canal of Hering, ductules, and ductular reactions, suggesting the involvement of stem/progenitor cells in cholangiocarcinoma development. To clarify the involvement of liver stem/progenitor cells in cholangiocarcinoma, we examined several stem/progenitor cell markers (CD133, CD44, OV6, and Oct3/4) in cholangiocarcinoma tissues analyzed by immunohistochemical staining, and measured 8-oxodG levels by using HPLC-ECD as an inflammation-related DNA lesion. In addition, a stem/progenitor cell factor Bmi1, 8-nitroguanine (formed during nitrative DNA damage), DNA damage response (DDR) proteins (phosphorylated ATM and γ-H2AX), and manganese-SOD (Mn-SOD) were analyzed by immunohistochemistry. Stem/progenitor cell markers (CD133, OV6, CD44, and Oct3/4) were positively stained in 56, 38, 47, and 56% of 34 cholangiocarcinoma cases, respectively. Quantitative analysis of 8-oxodG revealed significantly increased levels in CD133- and/or Oct3/4-positive tumor tissues compared to negative tumor tissues, as well as 8-nitroguanine formation detected by immunohistochemistry. In the cases of CD44- and/or OV6-positive tissue, no significant difference was observed. Cholangiocarcinoma patients with CD133- and/or Oct3/4-positive tumor tissues showed significantly lower expression of Mn-SOD and higher DDR protein, γ-H2AX. Moreover, CD133- and/or Oct3/4-positive cholangiocarcinoma patients had significant associations with tumor histology types, tumor stage, and poor prognoses. Our results suggest that CD133 and Oct3/4 in cholangiocarcinoma are associated with increased formation of DNA lesions and the DDR protein, which may be involved in genetic instability and lead to cholangiocarcinoma development with aggressive clinical features.

Barrett's esophagus (BE), an inflammatory disease, is a risk factor for Barrett's esophageal adenocarcinoma (BEA). Treatment of BE patients with proton pump inhibitors (PPIs) is expected to reduce the risk of BEA. We performed an immunohistochemical study to examine the formation of nitrative and oxidative DNA lesions, 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxygaunosine (8-oxodG), in normal esophageal, BE with pre- and post-treatment by PPIs and BEA tissues. We also observed the expression of an oxidant-generating enzyme (iNOS) and its transcription factor NF-κB, an antioxidant enzyme (Mn-SOD), its transcription factor (Nrf2) and an Nrf2 inhibitor (Keap1). The immunoreactivity of DNA lesions was significantly higher in the order of BEA>BE>normal tissues. iNOS expression was significantly higher in the order of BEA>BE>normal tissues, while Mn-SOD expression was significantly lower in the order of BEA

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N-acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using (32)P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H(2)O(2) with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.

Modification of DNA is believed to be a key step in mutagenesis and carcinogenesis. Reactive oxygen species (ROS) generated by environmental factors can cause oxidative DNA damage. This review focused on the role of oxidative DNA damage in mutagenesis and carcinogenesis mediated by environmental factors. This research investigated the mechanism of DNA damage induced by environmental chemicals and dietary factors using 32P-labeled DNA fragments obtained from the c-Ha-RAS-1 protooncogene and the p16 and p53 tumor suppressor genes. In addition, the content of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) was measured by using a high performance liquid chromatograph equipped with an electrochemical detector. 8-oxodG is probably one of the most abundant DNA lesion formed during oxidative stress. Antioxidants are considered as the most promising chemopreventive agents against various human cancers. However, some antioxidants play paradoxical roles acting as “double-edged sword”. The present research also investigated the mechanism of DNA damage induced by antioxidants using human cultured cell lines and 32P-labeled DNA fragments. This review shows recent experimental results and discusses the mechanisms of oxidative DNA damage in relation to carcinogenesis.

6-Hydroxydopamine (6-OHDA) is a neurotoxin to produce an animal model of Parkinson's disease. 6-OHDA increased the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), a biomarker of oxidatively damaged DNA, and induced apoptosis in human neuroblastoma SH-SY5Y cells. Iron or copper chelators inhibited 6-OHDA-induced 8-oxodG formation and apoptosis. Thus, iron and copper are involved in the intracellular oxidatively generated damage to DNA, a stimulus for initiating apoptosis. This study examined DNA damage caused by 6-OHDA plus metal ions using (32)P-5'-end-labelled DNA fragments. 6-OHDA increased levels of oxidatively damaged DNA in the presence of Fe(III)EDTA or Cu(II). Cu(II)-mediated DNA damage was stronger than Fe(III)-mediated DNA damage. The spectrophotometric detection of p-quinone and the scopoletin method showed that Cu(II) more effectively accelerated the 6-OHDA auto-oxidation and H(2)O(2) generation than Fe(III)EDTA. This study suggests that copper, as well as iron, may play an important role in 6-OHDA-induced neuronal cell death.

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD(+)-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H(2)O(2) and examined DNA cleavage, depletion of intracellular NAD(+), expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated beta-galactosidase (SA-beta-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H(2)O(2), though no cell death was observed. NAD(+) levels in TIG-3 cells treated with H(2)O(2) were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD(+) levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H(2)O(2) treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H(2)O(2) induced cell cycle arrest at 24h and increased SA-beta-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.

Chloramphenicol (CAP) was an old antimicrobial agent. However, the use of CAP is limited because of its harmful side effects, such as leukemia. The molecular mechanism through which CAP has been strongly correlated with leukemogenesis is still unclear. To elucidate the mechanism of genotoxicity, we examined DNA damage by CAP and its metabolites, nitroso-CAP (CAP-NO), N-hydroxy-CAP (CAP-NHOH), using isolated DNA. CAP-NHOH have the ability of DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in the presence of Cu(II), which was greatly enhanced by the addition of an endogenous reductant NADH. CAP-NO caused DNA damage in the presence of Cu(II), only when reduced by NADH. NADH can non-enzymatically reduce the nitroso form to hydronitroxide radicals, resulting in enhanced generation of reactive oxygen species followed by DNA damage through the redox cycle. Furthermore, we also studied the site specificity of base lesions in DNA treated with piperidine or formamidopyrimidine-DNA glycosylase, using (32)P-5'-end-labeled DNA fragments obtained from the human tumor suppressor gene. CAP metabolites preferentially caused double base lesion, the G and C of the ACG sequence complementary to codon 273 of the p53 gene, in the presence of NADH and Cu(II). Therefore, we conclude that oxidative double base lesion may play a role in carcinogenicity of CAP.

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-kappaB (NF-kappaB), 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

4-Hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus, is carcinogenic to rodents. To clarify the mechanism of carcinogenesis, we investigated DNA damage by 4-hydrazinobenzoic acid using (32)P-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes. 4-Hydrazinobenzoic acid induced Cu(II)-dependent DNA damage especially piperidine-labile formation at thymine and cytosine residues. Typical hydroxyl radical scavengers showed no inhibitory effects on Cu(II)-mediated DNA damage by 4-hydrazinobenzoic acid. Bathocuproine and catalase inhibited the DNA damage, indicating the participation of Cu(I) and H(2)O(2) in the DNA damage. These findings suggest that H(2)O(2) generated by the autoxidation of 4-hydrazinobenzoic acid reacts with Cu(I) to form reactive oxygen species, capable of causing DNA damage. Interestingly, catalase did not completely inhibit DNA damage caused by a high concentration of 4-hydrazinobenzoic acid (over 50 microM) in the presence of Cu(II). 4-Hydrazinobenzoic acid induced piperidine-labile sites frequently at adenine and guanine residues in the presence of catalase. 4-Hydrazinobenzoic acid increased formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in calf thymus DNA, whereas 4-hydrazinobenzoic acid did not increase the formation of 8-oxodG in the presence of catalase. ESR spin-trapping experiments showed that the phenyl radical was formed during the reaction of 4-hydrazinobenzoic acid in the presence of Cu(II) and catalase. Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF/mass) spectrometry analysis showed that phenyl radical formed adduct with adenosine and guanosine. These results suggested that 4-hydrazinobenzoic acid induced DNA damage via not only H(2)O(2) production but also phenyl radical production. This study suggests that both oxidative DNA damage and DNA adduct formation play important roles in the expression of carcinogenesis of 4-hydrazinobenzoic acid.

Antioxidants are considered as the most promising chemopreventive agents against various human cancers. However, some antioxidants play paradoxical roles, acting as "double-edged sword." A primary property of effective and acceptable chemopreventive agents should be freedom from toxic effects in healthy population. Miscarriage of the intervention by beta-carotene made us realize the necessity for evaluation of safety before recommending use of antioxidant supplements for chemoprevention. We have evaluated the safety of antioxidants on the basis of reactivity with DNA. Our results revealed that phytic acid, luteolin, and retinoic acid did not cause DNA damage under the experimental condition. Furthermore, phytic acid inhibited the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, in cultured cells treated with a H(2)O(2)-generating system. Thus, it is expected that these chemopreventive agents can safely protect humans against cancer. On the other hand, some chemopreventive agents with prooxidant properties (alpha-tocopherol, quercetin, catechins, isothiocyanates, N-acetylcysteine) caused DNA damage via generation of reactive oxygen species in the presence of metal ions and endogenous reductants under some circumstances. Furthermore, other chemopreventive agents (beta-carotene, genistein, daidzein, propyl gallate, curcumin) exerted prooxidant properties after metabolic activation. Therefore, further studies on safety should be required when antioxidants are used for cancer prevention.

Background/Aims Nucleic acid damage by reactive nitrogen and oxygen species may contribute to inflammation-related carcinogenesis. To investigate the extent of nucleic acid damage in hepatitis C virus infection and its change after interferon treatment, we measured 8-nitroguanine and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the liver of patients with chronic hepatitis C (CHC) before and after interferon therapy. Methods Hepatic accumulation of 8-nitroguanine and 8-OHdG was immunohistochemically evaluated in 20 CHC patients and 7 control patients with non-alcoholic fatty liver. Results Immunoreactivities of 8-nitroguanine and 8-OHdG were strongly detected in the liver from patients with CHC, but not in control livers. 8-Nitroguanine accumulation was found not only in infiltrating inflammatory cells, but also hepatocytes particularly in the periportal area. The accumulation of 8-nitroguanine and 8-OHdG increased with inflammatory grade (8-nitroguanine; P =0.0019, 8-OHdG; P =0.0009). In the sustained virological responder group after interferon therapy, 8-nitroguanine and 8-OHdG accumulation were markedly decreased in the liver (8-nitroguanine; P =0.018, 8-OHdG; P =0.018). Conclusions In this study, we demonstrated for the first time that 8-nitroguanine accumulated in the liver of patients with CHC. 8-Nitroguanine is a useful biomarker to evaluate the severity of HCV-induced chronic inflammation in relation to hepatocellular carcinoma.

Eugenol used as a flavor has potential carcinogenicity. DNA adduct formation via 2,3-epoxidation pathway has been thought to be a major mechanism of DNA damage by carcinogenic allylbenzene analogs including eugenol. We examined whether eugenol can induce oxidative DNA damage in the presence of cytochrome P450 using [ 32 P]-5′-end-labeled DNA fragments obtained from human genes relevant to cancer. Eugenol induced Cu(II)-mediated DNA damage in the presence of cytochrome P450 (CYP)1A1, 1A2, 2C9, 2D6, or 2E1. CYP2D6 mediated eugenol-dependent DNA damage most efficiently. Piperidine and formamidopyrimidine-DNA glycosylase treatment induced cleavage sites mainly at T and G residues of the 5′-TG-3′ sequence, respectively. Interestingly, CYP2D6-treated eugenol strongly damaged C and G of the 5′-ACG-3′ sequence complementary to codon 273 of the p53 gene. These results suggest that CYP2D6-treated eugenol can cause double base lesions. DNA damage was inhibited by both catalase and bathocuproine, suggesting that H 2 O 2 and Cu(I) are involved. These results suggest that Cu(I)–hydroperoxo complex is primary reactive species causing DNA damage. Formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine was significantly increased by CYP2D6-treated eugenol in the presence of Cu(II). Time-of-flight-mass spectrometry demonstrated that CYP2D6 catalyzed O -demethylation of eugenol to produce hydroxychavicol, capable of causing DNA damage. Therefore, it is concluded that eugenol may express carcinogenicity through oxidative DNA damage by its metabolite.

Inflammation mediated by infection is an important factor causing carcinogenesis. Opisthorchis viverrini (OV) infection is a risk factor of cholangiocarcinoma (CHCA), probably through chronic inflammation. Formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) were assessed in the liver of hamsters infected with OV. We newly produced specific anti-8-nitroguanine antibody without cross-reaction. Double immunofluorescence staining revealed that 8-oxodG and 8-nitroguanine were formed mainly in the same inflammatory cells and epithelium of bile ducts from day 7 and showed the strongest immunoreactivity on days 21 and 30, respectively. It is noteworthy that 8-oxodG and 8-nitroguanine still remained in epithelium of bile ducts on day 180, although amount of alanine aminotransferase activity returned to normal level. A time course of 8-nitroguanine was associated with iNOS expression. Furthermore, this study demonstrated that HO-1 expression and subsequent iron accumulation may be involved in enhancement of oxidative DNA damage in epithelium of small bile ducts. In conclusion, nitrative and oxidative DNA damage via iNOS expression in hamsters infected with OV may participate in CHCA carcinogenesis.

Interstrand DNA cross-linking has been considered to be the primary action mechanism of cyclophosphamide (CP) and its hydroperoxide derivative, 4-hydroperoxycyclophosphamide (4-HC). To clarify the mechanism of anti-tumor effects by 4-HC, we investigated DNA damage in a human leukemia cell line, HL-60, and its H(2)O(2)-resistant clone HP100. Apoptosis DNA ladder formation was detected in HL-60 cells treated with 4-HC, whereas it was not observed in HP100 cells. 4-HC significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, a marker of oxidative DNA damage, in HL-60 cells. On the other hand, CP did not significantly induce 8-oxodG formation and apoptosis in HL-60 cells under the same conditions as did 4-HC. Using (32)P-labeled DNA fragments from the human p53 tumor suppressor gene, 4-HC was found to cause Cu(II)-mediated oxidative DNA damage, but CP did not. Catalase inhibited 4-HC-induced DNA damage, including 8-oxodG formation, suggesting the involvement of H(2)O(2). The generation of H(2)O(2) during 4-HC degradation was ascertained by procedures using scopoletin and potassium iodide. We conclude that, in addition to DNA cross-linking, oxidative DNA damage through H(2)O(2) generation may participate in the anti-tumor effects of 4-HC.

In this study, we compared the DNA-damaging ability of bifunctional busulfan and monofunctional ethyl methanesulfonate (EMS, CH3SO2OC2H5) and their cytotoxicity towards human promyelocytic leukemia HL-60 cells. We also investigated the site specificity of busulfan-induced DNA damage using 32 P-5′end-labeled DNA fragments obtained from the human p16 tumor suppressor gene. Furthermore, time-of-flight mass spectrometry (TOF-MS) was performed to identify the DNA crosslinks formed by busulfan. Materials and Methods Materials. Restriction enzymes (EcoRI and BssHII) and proteinase K were from Boehringer Mannheim GmbH (Mannheim, Germany). T4 polynucleotide kinase was obtained from New England Biolabs (Beverly, MA). [γ- 32 P]ATP (222 TBq/mmol) was from New England Nuclear (Boston, MA). Diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) was from Dojin Chemical Co. (Kumamoto). Busulfan, calf thymus DNA, 3-hydroxypicolinic acid and citric acid were from Sigma Chemical Co. (St. Louis, MO). Acrylamide, dimethylsulfoxide, bisacrylamide and piperidine were from Wako Pure Chemical Industries (Osaka). EMS and ethanol were from Nacalai Tesque, Inc. (Kyoto). Mouse 3-methyladenine DNA glycosylase (Aag) was obtained from Trevigen Inc. (Gaithersburg, MD). Growth-inhibitory and cytotoxic effects of busulfan and EMS on cultured cells. HL-60 cells (2.5×10 5 cells/ml) were incubated with 100 or 200 µM busulfan or 200 µM EMS in 2 ml of RPMI 1640 (Gibco Laboratories, NY) supplemented with 6% fetal calf serum (FCS, Whittaker Bioproducts) for 24 h at 37°C. Cell viability was determined by trypan blue exclusion and counting in a hemocytometer. For statistical analysis of the data, an independent t test was used, and the criterion of significance was set at P

Green tea catechins, especially (-)-epigallocatechin gallate (EGCG), are believed to mediate much of the cancer chemopreventive effects of tea. However, it was reported that green tea catechins enhanced colon carcinogenesis in rats. Experiments using 32P-labeled DNA fragments obtained from human cancer-related genes showed that catechins induced DNA damage in the presence of metals such as Cu(II) and Fe(III) complexes. In the presence of Fe(III)EDTA, the order of DNA damaging ability was EGCG approximately (-)-epigallocatechin>(-)-epicatechin gallate>>catechin. Catechins plus Fe(III)EDTA caused DNA damage at every nucleotide, most likely due to *OH generation from H(2)O(2). In the presence of Cu(II), the order was (-)-epigallocatechin>catechin>EGCG>(-)-epicatechin gallate. Cu(II)-mediated DNA damage by EGCG occurred most frequently at T and G residues, especially of 5'-TG-3' and GG sequences. Catalase and bathocuproine inhibited the Cu(II)-mediated DNA damage, suggesting the involvement of H(2)O(2) and Cu(I). In the presence of metal ions, increased amounts of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were found in DNA treated with EGCG. Furthermore, EGCG increased amounts of 8-oxodG in HL-60 cells, but not in the H(2)O(2)-resistant clone HP100. When GSH was reduced by L-buthionine-[S, R]-sulfoximine, a low concentration of EGCG increased amounts of 8-oxodG in HL-60 cells, further supporting the involvement of H(2)O(2) in cellular DNA damage. It is concluded that EGCG can induce H(2)O(2) generation and subsequent damage to isolated and cellular DNA, and that oxidative DNA damage may mediate the potential carcinogenicity of EGCG.

Benz[a]anthracene (BA) is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) that are ubiquitous environmental pollutants. PAH carcinogenesis is explained by DNA adduct formation by PAH diol epoxide and oxidative DNA damage by PAH o-quinone. Benz[a]anthracene-trans-3,4-dihydrodiol (BA-3,4-dihydrodiol) is a minor metabolite but shows higher mutagenicity and tumorigenicity than parent BA. We confirmed that a BA o-quinone type metabolite, benz[a]anthracene-3,4-dione (BA-3,4-dione), induced oxidative DNA damage in the presence of cytochrome P450 reductase. Interestingly, we found that BA-3,4-dihydrodiol nonenzymatically caused Cu(II)-mediated DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine formation and the addition of NADH enhanced DNA damage. BA-3,4-dihydrodiol induced a double-base lesion of C and G at the 5'-ACG-3' sequence complementary to codon 273 of the human p53 tumor suppressor gene, which is known as a hotspot. The DNA damage was inhibited by catalase and bathocuproine, indicating the involvement of H(2)O(2) and Cu(I). Time-of-flight mass spectroscopic study suggested that BA-3,4-dihydrodiol undergoes Cu(II)-mediated autoxidation leading to the formation of its hydroxylated form of BA-3,4-dihydrodiol, capable of causing oxidative DNA damage. It is noteworthy that BA-3,4-dihydrodiol can nonenzymatically induce DNA damage more efficiently than BA-3,4-dione with metabolic activation. In conclusion, oxidative DNA damage induced by BA-3,4-dihydrodiol not only via quinone-type redox cycle but also via a new type of redox cycle participates in the expression of carcinogenicity of BA and BA-3,4-dihydrodiol.

3′-Azido-3′-deoxythymidine (AZT) is carcinogenic to experimental animals and can cause the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in humans and animals. To clarify the mechanism of carcinogenesis by AZT, we investigated DNA damage induced by its photodegradation products, using 32P-5′-end-labeled DNA fragments obtained from human genes. Following exposure to UVB, AZT induced DNA damage in the presence of Cu(II). Catalase inhibited DNA damage, indicating the involvement of H2O2. UVB-exposed AZT plus Cu(II) induced 8-oxodG formation in a dose-dependent manner. Mass spectrum of UVB-exposed AZT demonstrated the generation of a hydroxylamine derivative. The colorimetric determination suggested that AZT was converted into the hydroxylamine derivative depending on UVB doses. UVB-exposed AZT induced double base damage at the 5′-A CG -3′ sequence, complementary to a hot spot of the p53 gene. The basic compound, hydroxylamine, showed similar site specificity. The hydroxylamine derivative produced by photodegradation and/or possible metabolism of AZT induces oxidative DNA damage, which may participate in carcinogenesis.

Carcinogenic urethane (ethyl carbamate) forms DNA adduct via epoxide, whereas carcinogenic methyl carbamate can not. To clarify a mechanism independent of DNA adduct formation, we examined DNA damage induced by N-hydroxyurethane, a urethane metabolite, using 32P-5'-end-labeled DNA fragments. N-hydroxyurethane induced Cu(II)-mediated DNA damage especially at thymine and cytosine residues. DNA damage was inhibited by both catalase and bathocuproine, suggesting a role for H(2)O(2) and Cu(I) in DNA damage. Free (*) OH scavengers did not inhibit the DNA damage, although methional did inhibit it. These results suggest that reactive species, such as the Cu(I)-hydroperoxo complex, cause DNA damage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was increased by N-hydroxyurethane in the presence of Cu(II). When treated with esterase, N-hydroxyurethane induced 8-oxodG formation to a similar extent as that induced by hydroxylamine. Enhancement of DNA cleavages by endonuclease IV suggests that hydroxylamine induced depurination. Furthermore, hydroxylamine induced a significant increase in 8-oxodG formation in HL-60 cells but not in its H(2)O(2)-resistant clone HP 100 cells. o-Phenanthroline significantly inhibited the 8-oxodG formation in HL-60 cells, confirming the involvement of metal ions in the 8-oxodG formation by hydroxylamine. Electron spin resonance spectroscopy, utilizing Fe[N-(dithiocarboxy)sarcosine](3), demonstrated that nitric oxide (NO) was generated from hydroxylamine and esterase-treated N-hydroxyurethane. It is concluded that urethane may induce carcinogenesis through oxidation and, to a lesser extent, depurination of DNA by its metabolites.

We reviewed the mechanism of oxidative DNA damage with reference to metal carcinogenesis and metal-mediated chemical carcinogenesis. On the basis of the finding that chromium (VI) induced oxidative DNA damage in the presence of hydrogen peroxide (H2O2), we proposed the hypothesis that endogenous reactive oxygen species play a role in metal carcinogenesis. Since then, we have reported that various metal compounds, such as cobalt, nickel, and ferric nitrilotriacetate, directly cause site-specific DNA damage in the presence of H2O2. We also found that carcinogenic metals could cause DNA damage through indirect mechanisms. Certain nickel compounds induced oxidative DNA damage in rat lungs through inflammation. Endogenous metals, copper and iron, catalyzed ROS generation from various organic carcinogens, resulting in oxidative DNA damage. Polynuclear compounds, such as 4-aminobiphenyl and heterocyclic amines, appear to induce cancer mainly through DNA adduct formation, although their N-hydroxy and nitroso metabolites can also cause oxidative DNA damage. On the other hand, mononuclear compounds, such as benzene metabolites, caffeic acid, and o-toluidine, should express their carcionogenicity through oxidative DNA damage. Metabolites of certain carcinogens efficiently caused oxidative DNA damage by forming NADH-dependent redox cycles. These findings suggest that metal-mediated oxidative DNA damage plays important roles in chemical carcinogenesis.

We previously demonstrated that melatonin could be used as a chemopreventive agent for inhibiting cholangiocarcinoma (CCA) development in a hamster model. However, the cytotoxic activity of melatonin in cancer remains unclear. In the present study, we investigated the effect of melatonin on CCA cell lines. Human CCA cell lines (KKU-M055 and KKU-M214) were treated with melatonin at concentrations of 0.5, 1 and 2 mM for 48 h. Melatonin treatment exerted a cytotoxic effect on CCA cells by inhibiting CCA cell viability in a concentration-dependent manner. Treatment with melatonin, especially at 2 mM, increased intracellular reactive oxygen species (ROS) production and in turn led to increased oxidative DNA damage and 8-oxodG formation. Moreover, melatonin treatment enhanced the production of cytochrome c leading to apoptosis in a concentration-dependent manner, as indicated by increased expression of apoptosis-related proteins caspase-3 and caspase-7. In conclusion, melatonin acts as a pro-oxidant by activating ROS-dependent DNA damage and thus leading to the apoptosis of CCA cells.

The mechanism of DNA damage photosensitized by Nile blue (NB) was studied using (32)P-5'-end-labeled DNA fragments. NB bound to the DNA strand was possibly intercalated through an electrostatic interaction. Photoirradiated NB caused DNA cleavage at guanine residues when the DNA fragments were treated with piperidine. Consecutive guanines, the underlined G in 5'-GG and 5'-GGG, were selectively damaged through photoinduced electron transfer. The fluorescence lifetime of NB was decreased by guanine-containing DNA sequence, supporting this mechanism. Single guanines were also slightly damaged by photoexcited NB, and DNA photodamage by NB was slightly enhanced in D2O. These results suggest that the singlet oxygen mechanism also partly contributes to DNA photodamage by NB. DNA damage photosensitized by NB via electron transfer may be an important mechanism in medicinal applications of photosensitizers, such as photodynamic therapy in low oxygen.

We examined the redox properties of the "carcinogenic" catechol and the "noncarcinogenic" hydroquinone in relation to different DNA damaging activities and carcinogenicity using 32P-labeled DNA fragments obtained from the human genes. In the presence of endogenous NADH and Cu2+, catechol induces stronger DNA damage than hydroquinone, although the magnitudes of their DNA damaging activities were reversed in the absence of NADH. In both cases, DNA damage resulted from base modification at guanine and thymine residues in addition to strand breakage induced by Cu+ and H2O2, generated during the oxidation of catechol and hydroquinone into 1,2-benzoquinone and 1,4-benzoquinone, respectively. EPR and 1H NMR studies indicated that 1,2-benzoquinone is converted directly into catechol through a nonenzymatic two-electron reduction by NADH whereas 1,4-benzoquinone is reduced into hydroquinone through a semiquinone radical intermediate through two cycles of one-electron reduction. The reduction of 1,2-benzoquinone by NADH proceeds more rapidly than that of 1,4-benzoquinone. This study demonstrates that the rapid 1,2-benzoquinone two-electron reduction accelerates the redox reaction turnover between catechol and 1,2-benzoquinone, resulting in the enhancement of DNA damage. These results suggest that the differences in NADH-mediated redox properties of catechol and hydroquinone contribute to their different carcinogenicities.

Reactive species generated by chemicals and UV radiation can cause sequence-specific DNA damage and play important roles in mutagenesis, carcinogenesis and aging. We have investigated sequence specificity of oxidative stress-mediated DNA damage by using 32P-labeled DNA fragments obtained from the human c-Ha-ras-1 and p53 genes. Free hydroxyl radical causes DNA damage with no marked site specificity. Reactive nitrogen species, sulfate radicals, nitrogen-centered radicals, benzoyloxyl radical and alkoxyl radical show different sequence specificity. Benzoyloxyl radical specifically causes damage to the 5'-G in GG sequence. UVA radiation also causes DNA damage at this site through electron transfer in the presence of certain photosensitizers. The 5'-G in GG sequence is easily oxidized because a large part of the highest occupied molecular orbital is distributed on this site. On the basis of these findings, the sequence specificity of DNA damage is presumably determined by (a) redox potential of reactive species; (b) ionization potential of DNA bases; and (c) site-specific binding of metal ion to DNA. Here we discuss the mechanisms of sequence-specific DNA damage in relation to carcinogenesis and aging.

Hydroxyurea is a chemotherapeutic agent used for the treatment of myeloproliferative disorders (MPD) and solid tumors. The mutagenic and carcinogenic potential of hydroxyurea has not been established, although hydroxyurea has been associated with an increased risk of leukemia in MPD patients. To clarify whether hydroxyurea has potential carcinogenicity, we examined site-specific DNA damage induced by hydroxyurea using (32)P-5'-end-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c-Ha-ras-1 protooncogene. Hydroxyurea caused Cu(II)-mediated DNA damage especially at thymine and cytosine residues. NADH efficiently enhanced hydroxyurea-induced DNA damage. The DNA damage was almost entirely inhibited by catalase and bathocuproine, a Cu(I)-specific chelator, suggesting the involvement of hydrogen peroxide (H(2)O(2)) and Cu(I). Typical free hydroxyl radical scavengers did not inhibit DNA damage by hydroxyurea, but methional did. These results suggest that crypto-hydroxyl radicals such as Cu(I)-hydroperoxo complex (Cu(I)-OOH) cause DNA damage. Formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was induced by hydroxyurea in the presence of Cu(II). An electron spin resonance spectroscopic study using N-(dithiocarboxy)sarcosine as a nitric oxide (NO)-trapping reagent demonstrated that NO was generated from hydroxyurea in the presence and absence of catalase. In addition, the generation of formamide was detected by both gas chromatography-mass spectrometry (GC-MS) and time-of-flight-mass spectrometry (TOF-MS). A high concentration of hydroxyurea induced depurination at DNA bases in an H(2)O(2)-independent manner, and endonuclease IV treatment led to chain cleavages. These results suggest that hydroxyurea could induce base oxidation as the major pathway of DNA modification and depurination as a minor pathway. Therefore, it is considered that DNA damage by hydroxyurea participates in not only anti-cancer activity, but also carcinogenesis.

An important approach to improve chemotherapy of members of the bleomycin (BLM) family of antibiotics is to find compounds (amplifiers) that enhance the activity of BLM-mediated DNA cleavage and apoptosis. Using a DNA-sequencing technique and pulsed field gel electrophoresis, we have investigated whether BLM-mediated cleavage of isolated and cellular DNA is amplifed by three compounds (RW-12, LS-20, 1S-5Me) which have a conformationally flexible, unfused polyaromatic system and cationic side chain in the molecules. RW-12 enhanced most effectively both pepleomycin (PEM)-induced cytotoxicity and apoptosis. The order of the maximum enhancing effect of amplifiers on PEM-mediated DNA damage is RW-12 > LS-20 > 1S-5Me. RW-12 amplified PEM-mediated DNA cleavage most effectively not only in vitro but also in cultured cells. We have reported that the order of the DNA binding constants of these compounds is RW-12 > LS-20 > 1S-5Me. In this study, we found a good correlation between PEM-mediated cleavage of isolated DNA and cellular DNA. These results suggest that BLM amplifiers bind to DNA and by doing so enhance drug-mediated DNA degradation, ultimately leading to apoptosis. The present study on amplifiers of anticancer agents shows a novel approach to the potentially effective anticancer therapy.

Nitroxyl anion (NO−), the one-electron reduction product of nitric oxide (NO⋅), is formed under various physiological conditions. We have used four different assays (DNA strand breakage, 8-oxo-deoxyguanosine formation in calf thymus DNA, malondialdehyde generation from 2′-deoxyribose, and analysis of site-specific DNA damage using32P-5′-end-labeled DNA fragments of the human p53tumor suppressor gene and the c-Ha-ras-1 protooncogene) to study the effects of NO− generated from Angeli's salt on DNA damage. It was found that strong oxidants are generated from NO−, especially in the presence of H2O2 plus Fe(III)-EDTA or Cu(II). NO⋅released from diethylamine-NONOate had no such effect. Distinct effects of hydroxyl radical (HO⋅) scavengers and patterns of site-specific DNA cleavage caused by Angeli's salt alone or by Angeli's salt, H2O2 plus metal ion suggest that NO− acts as a reductant to catalyze the formation of the HO⋅ from H2O2 plus Fe(III) and formation of Cu(I)-peroxide complexes with a reactivity similar to HO⋅ from H2O2 and Cu(II). Angeli's salt and H2O2 exerted synergistically cytotoxic effects to MCF-7 cells, determined by lactate dehydrogenase release assay. Thus NO− may play an important role in the etiology of various pathophysiological conditions such as inflammation and neurodegenerative diseases, especially when H2O2 and transition metallic ions are present.

Pulsed field gel electrophoresis showed that the initiation time of DNA breakage induced by the DNA alkylating agent duocarmycin A, which is not a redox-cycling agent, was almost the same in the human leukemia cell line HL-60 and its H2O2-resistant clone HP100. Catalase activity of HP100 cells was much higher than that of HL-60 cells. Duocarmycin A-mediated DNA ladder formation in HP100 cells was delayed compared with that in HL-60 cells, suggesting the involvement of H2O2 in duocarmycin A-induced apoptosis. Flow cytometry demonstrated that peroxide formation preceded loss of mitochondrial membrane potential (delta psi m) in cells treated with duocarmycin A. Then, caspase-3 was activated, followed by DNA ladder formation. These findings suggest that DNA damage by duocarmycin A induces H2O2 generation, which causes delta psi m loss and subsequently caspase-3 activation, resulting in apoptosis.

DNA damage by metabolites of a food additive, butylated hydroxytoluene (BHT), was investigated as a potential mechanism of carcinogenicity. The mechanism of DNA damage by 2,6-di-tert-butyl-p-benzoquinone (BHT-quinone), 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone (BHT-OOH), and 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHT-CHO) in the presence of metal ions was investigated by using 32P-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. BHT-OOH caused DNA damage in the presence of Cu(II), whereas BHT-quinone and BHT-CHO did not. However, BHT-quinone did induce DNA damage in the presence of NADH and Cu(II). Bathocuproine inhibited Cu(II)-mediated DNA damage, indicating the participation of Cu(I) in the process. Catalase also inhibited DNA damage induced by BHT-quinone, but not that induced by BHT-OOH. The DNA cleavage pattern observed with BHT-quinone plus NADH was different from that seen with BHT-OOH. With BHT-quinone plus NADH, piperidine-labile sites could be generated at nucleotides other than adenine residue. BHT-OOH caused cleavage specifically at guanine residues. Pulsed field gel electrophoresis showed that BHT-OOH and BHT-quinone induced DNA strand breaks in cultured cells, whereas BHT-CHO did not. Both BHT-quinone and BHT-OOH induced internucleosomal DNA fragmentation, which is the characteristic of apoptosis. Furthermore, flow cytometry analysis revealed an increase of peroxides in cultured cells treated with BHT-OOH or BHT-quinone. These results suggest that BHT-OOH participates in oxidative DNA damage directly, whereas BHT-quinone causes DNA damage through H2O2 generation, which leads to internucleosomal DNA fragmentation.

The ability of Cu(II) and Fe(III) to promote site-specific DNA damage in the presence of endogenous reductants was investigated by using 32P-5′-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Ascorbate induced metal-dependent DNA damage most efficiently (ascorbate>GSH>NADH). Cu(II) induced endogenous reductants-dependent DNA damage more efficiently than Fe(III). Endogenous reductants plus Fe(III) caused DNA cleavage at every nucleotide, without marked site preference. DNA damage by Fe(III) was inhibited by hydroxyl free radical (⋅OH) scavengers and catalase. These results suggest that endogenous reductants plus Fe(III) generate free or extremely near free ⋅OH via H2O2 formation, and that ⋅OH causes DNA damage. In the presence of 50 μM Cu(II) in bicarbonate buffer, ascorbate caused DNA cleavage frequently at sites of two or more adjacent guanine residues. In contrast, in the presence of 20 μM Cu(II), ascorbate caused DNA cleavage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited DNA damage by Cu(II), whereas ⋅OH scavengers did not. Fe(III)-dependent 8-oxo-7,8-dihydro-2′-deoxyguanosine formation was inhibited by ⋅OH scavengers, whereas no inhibition by ⋅OH scavengers was observed with Cu(II). These results suggest that ⋅OH is the main active species formed with Fe(III), whereas copper-peroxide complexes with a reactivity similar to ⋅OH participate in Cu(II)-dependent DNA damage. The polyguanosine sequence specificity of DNA damage in the presence of high concentrations of Cu(II) can be explained by the preferential binding of Cu(II) to guanine residues.

It was recently reported that p-aminophenol (p-AP) induces DNA cleavage in mouse lymphoma cells, CHO cells and human lymphoblastoid cells. The mutagenicity of p-AP has not, however, been detected by reverse mutation assays. The purpose of this study was to assess the mutagenicity of p-AP by reverse mutation assay using Escherichia coli WP2uvrA/pKM101, which has a spectrum for detecting mutations different from those of other strains in the family with an AT base pair at the mutation site and has higher sensitivity to certain oxidative mutagens as compared to other strains. We found that p-AP was mutagenic to E. coli WP2uvrA/pKM101. The mutagenic activity of this compound was suppressed with the addition of dimethylsulfoxide or catalase, suggesting the involvement of active oxygen species in the mutagenic process induced by p-AP. To further elucidate the underlying mechanism, we used isolated DNA for the following experiments. It was revealed, by gel electrophoretic analysis, that p-AP induced DNA cleavage in the presence of Fe(III). However, p-AP alone did not induce this cleavage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by p-AP in calf thymus DNA was also detected in the presence of Fe(III) by HPLC with an electrochemical detector. ESR-spin trapping experiments using DMPO detected the production of hydroxyl radical (.OH) in the solution of p-AP with Fe(III). Both p-AP mediated DNA damages and .OH production by p-AP in the presence of Fe(III) were completely inhibited by .OH scavengers (ethanol, mannitol, sodium formate, dimethylsulfoxide) and catalase. These results suggest that .OH derived from the reaction between H2O2 and Fe(III) (Fenton reaction) participates in the oxidative DNA damage. Accordingly, the same mechanism might be working in E. coli WP2uvrA/pKM101 during induction of the mutation by p-AP.

Two hair dye components, carcinogenic 4-nitro-2-aminophenol and 5-nitro-2-aminophenol, induced Cu(II)-dependent DNA cleavage frequently at thymine and guanine residues in DNA fragments obtained from the c-Ha-ras-1 protooncogene. When the p53 tumor suppressor gene was used, 4-nitro-2-aminophenol caused Cu(II)-dependent piperidine-labile sites at poly G sequences. In the presence of Cu(II), both components increased 8-oxo-7,8-dihydro-2′-deoxyguanosine formation in DNA. The inhibitory effects of catalase and bathocuproine on DNA damage suggest the involvement of H2O2 and Cu(I). It is speculated that nitro-2-aminophenols undergo Cu(II)-mediated autoxidation to generate active oxygen species causing DNA damage which leads to their carcinogenesis.

The tryptophan metabolites 3-hydroxyanthranilic acid (3-HAA) and 3-hydroxykynurenine (3-HKyn) are carcinogens. DNA damage by 3-HAA and 3-HKyn in the presence of metal ions was investigated as a potential mechanism of their carcinogenicity. Pulsed field gel electrophoresis showed that in the presence of Mn(II), 3-HAA and 3-HKyn induced DNA double-strand breaks in cultured human cells. DNA single-strand breaks were observed with alkali treatment. The enhancing effect of catalase inhibitor and the inhibitory effect of o-phenanthroline on the strand breakage indicated the involvement of H 2 O 2 and endogenous transition metal ion. Damage to DNA fragments obtained from c-Ha-ras-1 protooncogene was investigated by a DNA sequencing technique. 3-HAA and 3-HKyn induced piperidine-labile sites frequently at thymine and guanine residues in the presence of Cu(II). The inhibitory effects of bathocuproine and catalase on Cu(II)-mediated DNA damage suggest that Cu(I) and H 2 O 2 have important roles in the production of active species causing DNA damage. The Cu(II)-mediated DNA damage was enhanced by preincubation of 3-HAA with Mn(II). UV-visible spectroscopy showed that Mn(II) and Cu(II) enhanced the rate of autoxidation of 3-HAA in different ways. These results suggest that in the presence of Mn(II) or Cu(II), these tryptophan metabolites produce H 2 O 2 , which is activated by transition metal ion to cause damage to DNA both in the case of isolated DNA and cultured cells

The U.S. National Toxicology Program has shown clear evidence of carcinogenicity of nickel subsulfide (Ni(3)S(2)) and some evidence of carcinogenicity of NiO (green) in rats. In the present study, DNA damage in cultured cells and in lungs of rats induced by nickel compounds was investigated to clarify the mechanism of nickel carcinogenesis. In cultured HeLa cells, Ni(3)S(2) induced a significant increase in 8-hydroxydeoxyguanosine (8-OH-dG) formation, whereas NiO (black), NiO (green), and NiSO(4) did not. On the other hand, in rats, intratracheal instillation of all these nickel compounds significantly increased 8-OH-dG content in the lungs. The disparities in DNA damage between cultured cells and animals could be accounted for by two different mechanisms for nickel-induced oxidative DNA damage in lungs of rats. One is direct oxidative DNA damage: Ni(II) enters the cells and then reacts with endogenous and/or nickel sulfide-produced hydrogen peroxide (H(2)O(2)) to give reactive oxygen species that cause DNA damage. This mechanism is supported by oxidative damage to isolated DNA treated with Ni(II) and H(2)O(2). The other mechanism is indirect oxidative DNA damage due to inflammation. This double mechanism for DNA damage may explain the relatively high carcinogenic risk associated with Ni(3)S(2).

To investigate whether mutant stem cells participate in inflammation-related carcinogenesis, we performed immunohistochemical analysis to examine nitrative and oxidative DNA lesions (8-nitroguanine and 8-oxodG) and a stem cell marker Oct3/4 in bladder tissues obtained from cystitis and bladder cancer patients infected with Schistosomahaematobium (S. haematobium). We also detected the expression of nuclear factor-κB (NF-κB) and inducible nitric oxide synthase (iNOS), which lead to 8-nitroguanine formation. The staining intensity of 8-nitroguanine and 8-oxodG was significantly higher in bladder cancer and cystitis tissues than in normal tissues. iNOS expression was colocalized with NF-κB in 8-nitroguanine-positive tumor cells from bladder cancer patients. Oct3/4 expression was significantly increased in cells from S. haematobium-associated bladder cancer tissues in comparison to normal bladder and cancer tissues without infection. Oct3/4 was also expressed in epithelial cells of cystitis patients. Moreover, 8-nitroguanine was formed in Oct3/4-positive stem cells in S. haematobium-associated cystitis and cancer tissues. In conclusion, inflammation by S.haematobium infection may increase the number of mutant stem cells, in which iNOS-dependent DNA damage occurs via NF-κB activation, leading to tumor development.

Amitrole (3-amino-1,2,4-triazole) is a widely used herbicide. Amitrole induces thyroid and liver tumors in rodents. However, the mechanism of carcinogenesis by amitrole remains to be clarified. To clarify the mechanism of carcinogenesis induced by amitrole, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic of oxidatively generated DNA damage, by an amitrole metabolite, 3-amino-5-mercapto-1,2,4-triazole (AMT), in the presence of Cu(II). The amount of 8-oxodG was increased by AMT in the presence of Cu(II). AMT-induced 8-oxodG formation was enhanced in deuterium oxide (D₂O), which prolongs the half life of singlet oxygen (¹O₂), more than that in H₂O. Sodium azide and 1,4-diazabicyclo[2,2,2]-octane (DABCO), potent and relatively specific scavengers of ¹O₂, inhibited AMT-mediated 8-oxodG formation. Bathocuproine, a Cu(I) chelator, also inhibited the 8-oxodG formation. On the other hand, typical OH scavengers did not inhibit the generation of 8-oxodG. AMT plus Cu(II) also induced piperidine-labile DNA lesions frequently at every guanine residue. These results suggest that ¹O₂ and Cu(I) play an important role in DNA damage induced by AMT. It is concluded that oxidatively generated DNA damage induced by AMT via the generation of ¹O₂ may contribute to carcinogenicity of amitrole.

Cancer photodynamic therapy (PDT) requires photosensitizers that efficiently and selectively destroy tumor cells. We investigated 5,10,15,20-tetrakis (N-methyl-4-pyridyl)-21H,23H-porphyrin (TMPyP) as a potential cancer treatment. Confocal fluorescence microscopy showed that TMPyP was localized in the nuclei, whereas 5-aminolevulinic acid (ALA)-derived protoporphyrin IX (PPIX) was localized diffusely in the cytoplasm of human leukemia (HL-60) cells. In HL-60 cells under UVA irradiation, TMPyP effectively induced apoptosis. Moreover, 8-oxo-7,8-dihydro-2'-deoxyguanosine, an oxidative product of 2'-deoxyguanosine, was accumulated in the DNA of cells treated with photoirradiated TMPyP, whereas only small amounts were observed in ALA-treated cells in the presence of UVA light. TMPyP and UVA caused extensive damage at every guanine residue in DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 proto-oncogene, whereas PPIX induced little DNA damage under these conditions. Electron spin resonance spectroscopy using a singlet oxygen (1O2) probe and D2O showed that photoexcited TMPyP generated 1O2. These results suggest that photoexcited TMPyP reacts with oxygen to generate 1O2, which in turn, oxidizes guanine residues. Taken together, the results demonstrated that TMPyP was localized in the nucleus where it was photosensitized to induce DNA damage, suggesting that TMPyP may have clinical utility as a nucleus-targeted PDT.

Aspirin has been proposed as a possible chemopreventive agent. On the other hand, a recent cohort study showed that aspirin may increase the risk for pancreatic cancer. To clarify whether aspirin is potentially carcinogenic, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is correlated with the incidence of cancer, in cultured cells treated with 2,3-dihydroxybenzoic acid (2,3-DHBA), a metabolite of aspirin. 2,3-DHBA induced 8-oxodG formation in the PANC-1 human pancreatic cancer cell line. 2,3-DHBA-induced DNA single-strand breaks were also revealed by comet assay using PANC-1 cells. Flow cytometric analyses showed that 2,3-DHBA increased the levels of intracellular reactive oxygen species (ROS) in PANC-1 cells. The 8-oxodG formation and ROS generation were also observed in the HL-60 leukemia cell line, but not in the hydrogen peroxide (H(2)O(2))-resistant clone HP100 cells, suggesting the involvement of H(2)O(2). In addition, an hprt mutation assay supported the mutagenicity of 2,3-DHBA. We investigated the mechanism underlying the 2,3-DHBA-induced DNA damage using (32)P-labeled DNA fragments of human tumor suppressor genes. 2,3-DHBA induced DNA damage in the presence of Cu(II) and NADH. DNA damage induced by 2,3-DHBA was enhanced by the addition of histone peptide-6 [AKRHRK]. Interestingly, 2,3-DHBA and histone peptide-6 caused base damage in the 5'-ACG-3' and 5'-CCG-3' sequences, hotspots of the p53 gene. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Typical hydroxyl radical scavengers did not inhibit the DNA damage. These results suggest that ROS derived from the reaction of H(2)O(2) with Cu(I) participate in the DNA damage. In conclusion, 2,3-DHBA induces oxidative DNA damage and mutations, which may result in carcinogenesis.

Reactive nitrogen species are considered to participate in inflammation-related carcinogenesis through DNA damage. 8-Nitroguanine is a specific marker of inflammation-related carcinogenesis. Epstein-Barr virus infection-related nasopharyngeal carcinoma (NPC) is one of the most prevalent malignant tumors in southern China and Southeast Asia, and its prognosis has been poor for decades. Previously, we demonstrated that nitrative DNA damage, such as 8-nitroguanine formation, occurs in cancer cells of NPC patients (Ma et al. Int. J. Cancer 2008). In the present study, to investigate the involvement of stem cells in NPC, we performed immunohistochemical analyses to examine nitrative DNA lesions (8-nitroguanine) and several cancer stem / progenitor cell markers (CD44v6, CD24 and ALDH1A1) in nasopharyngeal tissues obtained from chronic nasopharyngitis and NPC patients. The staining intensity of 8-nitroguanine was significantly higher in cancer cells and inflammatory cells in stroma of NPC than in chronic nasopharyngitis tissues. Expression levels of CD44v6 and ALDH1A1 were significantly increased in cancer cells of primary NPC specimens in comparison to chronic nasopharyngitis tissues. 8-Nitroguanine was detected in CD44v6- or ALDH1A1-positive stem cells in NPC tissues. In the case of CD24 staining, there was no significant difference between NPC and chronic nasopharyngitis tissues. Intensive staining of CD44v6 and ALDH1A1 were also detected in NPC cell line HK1 compared to immortalized nasopharyngeal epithelial cells NP460 by a double immunofluorescence study and western blotting assay. In conclusion, CD44v6 and ALDH1A1 could be candidates of cancer stem cell markers for NPC. The present results indicate the possible mechanism by which inflammation causes NPC by inducing inflammatory processes and formation of 8-nitroguanine in CD44v6- and / or ALDH1A1-positive stem cells, and mutant stem cells participate in NPC development.

Although capsaicin exhibits antitumor activity, carcinogenic potential has also been reported. To clarify the mechanism for expression of potential carcinogenicity of capsaicin, we examined DNA damage induced by capsaicin in the presence of metal ion and various kinds of cytochrome P450 (CYP) using 32P-5'-end-labeled DNA fragments. Capsaicin induced Cu(II)-mediated DNA damage efficiently in the presence of CYP1A2 and partially in the presence of 2D6. CYP1A2-treated capsaicin caused double-base lesions at 5'-TG-3', 5'-GC-3' and CG of the 5'-ACG-3' sequence complementary to codon 273, a hotspot of p53 gene. DNA damage was inhibited by catalase and bathocuproine, a Cu(I) chelator, suggesting that reactive species derived from the reaction of H2O2 with Cu(I) participate in DNA damage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine was significantly increased by CYP1A2-treated capsaicin in the presence of Cu(II). Therefore, we conclude that Cu(II)-mediated oxidative DNA damage by CYP-treated capsaicin seems to be relevant for the expression of its carcinogenicity.

Tetrahydropapaveroline (THP), a metabolite of dopamine, has been suspected to be associated with dopaminergic neurotoxicity of L-DOPA. THP induced apoptosis in human leukemia cell line HL-60 cells, but did not in its hydrogen peroxide (H(2)O(2))-resistant clone HP100. THP-induced DNA ladder formation in HL-60 cells was inhibited by a metal chelator. THP induced damage to (32)P-labeled DNA fragments in the presence of metals. In the presence of Fe(III)EDTA, THP caused DNA damage at every nucleotide. The DNA damage was inhibited by free hydroxy radical ((.)OH) scavengers and catalase, suggesting that the Fe(III)EDTA-mediated DNA damage is mainly due to (.)OH generation. In the presence of Cu(II), THP caused DNA damage mainly at T and G of 5'-TG-3' sequence. The inhibitive effect of catalase and bathocuproine on Cu(II)-mediated DNA damage suggested that H(2)O(2) and Cu(I) participate in the DNA damage. This study demonstrated that THP-induced apoptosis via reactive oxygen species generated from reaction of H(2)O(2) and metals plays an important role in cytotoxicity of L-DOPA.