8-oxodG Formation Research Articles

酵母細胞におけるグアニン塩基の電気化学的測定法―紫外線照射影響研究への応用―

The cellular harm or mutation results from the indirect reaction through reactive oxygen species (ROS) generated by ultraviolet (UV) ray or direct reaction (such as cyclobutane pyrimidine dimmer (CPD) and 6-4 photoproduct). The amount of intracellular guanine nucleotide irradiated by the 0-80 J/m2 UV-C (254 nm) was measured by means of cyclic voltammetry (CV). The oxidation current peak of intracellular guanine nucleotide was observed. The current peak which reflects the concentration of intracellular guanine base decreased to about 80% of initial value with 80 J/m2 of UV-irradiation. The concentration of guanine nucleotide could be estimated from the oxidation peak current in CV. Moreover, formation of 8-oxodG was estimated by the increase in the mutation frequency from UV-irradiated cells of base excision repair deficient strain. It was shown that the decrease in the peak current is due to the oxidation of the guanine base. This analytical method is useful for the elucidation of the oxidation mechanism of the cell and biological tissues.

Damage to cellular and isolated DNA induced by a metabolite of aspirin

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 2O 2)-resistant clone HP100 cells, suggesting the involvement of H 2O 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 32P-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 2O 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.

Urinary 8-Oxo-7,8-Dihydro-2′-Deoxyguanosine in Patients with Parasite Infection and Effect of Antiparasitic Drug in Relation to Cholangiocarcinogenesis

Parasite infection of Opisthorchis viverrini is a major risk factor for cholangiocarcinoma. Our previous immunohistochemical studies showed that O. viverrini infection induced oxidative DNA lesions in the bile duct epithelium during cholangiocarcinoma development. The current study assessed the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an oxidative DNA lesion, in the urine and leukocytes of O. viverrini-infected subjects and cholangiocarcinoma patients. Forty-nine O. viverrini-infected patients, 55 cholangiocarcinoma patients, and 17 healthy controls were enrolled in the study. We measured 8-oxodG levels in the urine and leukocytes of these subjects using an electrochemical detector coupled to high-performance liquid chromatography. O. viverrini-infected patients were assessed before treatment and 2 months and 1 year after praziquantel treatment. Urinary 8-oxodG levels were significantly higher in cholangiocarcinoma patients (6.83 +/- 1.00 microg/g creatinine) than in O. viverrini-infected patients (4.45 +/- 0.25 mug/g creatinine; P < 0.05) and healthy subjects (3.03 +/- 0.24 microg/g creatinine; P < 0.01) and higher in O. viverrini-infected subjects than in healthy subjects (P < 0.01). The urinary 8-oxodG levels in O. viverrini-infected patients significantly decreased 2 months after praziquantel treatment and were comparable with levels in healthy subjects 1 year after treatment. Urinary 8-oxodG levels were significantly correlated with leukocyte 8-oxodG levels, plasma nitrate/nitrite levels, and aspartate aminotransferase activity. In conclusion, this study, in addition to our previous studies, indicates that 8-oxodG formation by parasite infection may play an important role in cholangiocarcinoma development. Urinary 8-oxodG may be a useful biomarker to monitor not only infection but also carcinogenesis.

Mechanism of metal-mediated DNA damage and apoptosis induced by 6-hydroxydopamine in neuroblastoma SH-SY5Y cells

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 32P-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 H2O2 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.

Prevention of artifactual oxidation in determination of cellular 8-oxo-7,8-dihydro-2′-deoxyguanosine by isotope-dilution LC–MS/MS with automated solid-phase extraction

A highly sensitive quantitative method based on LC–MS/MS was developed to directly measure 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 2′-deoxyguanosine (dG) in crude DNA hydrolysates. With the use of isotopic internal standards and online solid-phase extraction (SPE), this method has overcome the artifactual response often observed during electrospray ionization by optimizing the washing conditions of online SPE to remove excess dG and allows 8-oxodG and dG to be accurately and simultaneously monitored by mass spectrometry. The detection limit of this method was estimated as 1.8 fmol for 8-oxodG. With this method, we further investigated the artifactual oxidation that occurred during concentration and purification of the DNA hydrolysates, commonly used before sample analysis. Our results demonstrated that drying under vacuum or purification with C18 cartridges led to a significant increase in the measured 8-oxodG by 6.8–30 8-oxodG/10 6 dG. The artifactual formation of 8-oxodG can be reduced only by adding desferrioxamine (DFO) and not 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). However, DFO still failed to offer complete protection against oxidation during DNA hydrolysate concentration and purification. Therefore, to effectively prevent the artifacts formed during workup, the simplest approach is to use a direct measurement method involving an online enrichment/purification technique as proposed in this study.

Mechanism of UVA-dependent DNA Damage Induced by An Antitumor Drug Dacarbazine in Relation to its Photogenotoxicity

It has been reported that dacarbazine (DTIC) is photogenotoxic. The purpose of this study is to clarify the mechanism of photogenotoxicity induced by DTIC. We examined DNA damage induced by UVA-irradiated DTIC using 32P-5'-end-labeled DNA fragments obtained from human genes. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in calf thymus DNA was measured by high performance liquid chromatograph with an electrochemical detector. Electron spin resonance (ESR) spin-trapping experiments were performed to detect radical species generated from UVA-irradiated DTIC. UVA-irradiated DTIC caused DNA damage at guanine residues, especially at the 5'-GGT-3' sequence in the presence of Cu(II) and also induced 8-oxodG generation in calf thymus DNA. DTIC-induced photodamage to DNA fragments was partially inhibited by catalase, whereas 8-oxodG formation was significantly increased by catalase. NaN3, a carbene scavenger, inhibited DNA damage and 8-oxodG formation in a dose-dependent manner, suggesting that carbene intermediates are involved. The ESR spin-trapping experiments demonstrated the generation of aryl radicals in the process of photodegradation of DTIC. Photoactivated DTIC generates the carbene and aryl radicals, which may induce both DNA adduct and 8-oxodG formation, resulting in photogenotoxicity. This study could provide an insight into the safe usage of DTIC.

A possible photosensitizer: Tobacco-specific nitrosamine, 4-( N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), induced mutations, DNA strand breaks and oxidative and methylative damage with UVA

We discovered the directly acting mutagenicity of the tobacco-specific nitrosamine, 4-( N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), with UVA light (320–400 nm) in Ames bacteria and phage M13mp2 in the absence of metabolic activation. We have investigated the spectrum of mutations caused by UVA-activated NNK. The majority (57%) of induced sequence changes were comprised of GC to CG, GC to TA and GC to AT. This suggested that modification of guanine residues was responsible for these mutations. Hence, we explored the formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) and O 6-methylguanine (O 6meG) in the DNA. When calf thymus DNA was treated with NNK and UVA, the amount of 8-oxodG/dG and O 6meG/G in the DNA increased up to 20-fold and 100-fold, respectively, compared with the untreated control. DNA strand breaks were observed following NNK and UVA treatment, and the strand breaks were suppressed in the presence of scavengers for oxygen and NO radical. The formation of NO was also observed in NNK solutions irradiated with UVA. We analyzed the photodynamic spectrum of mutation induction, 8-oxodG formation and NO formation using monochromatic radiation. The patterns of the action spectra were comparable to the absorption spectrum of NNK. We conclude that NNK may act as a photosensitizer in response to UVA to produce NO and other oxidative and alkylative intermediates following the formation of 8-oxodG and O 6meG in DNA, which may lead to mutations and DNA strand breaks.

N-Hydroxy-4-(4-chlorophenyl)thiazole-2(3H)-thione as a Photochemical Hydroxyl-radical Source: Photochemistry and Oxidative Damage of DNA (Strand Breaks) and 2′-Deoxyguanosine (8-oxodG Formation)¶

N-Hydroxy-4-(4-chlorophenyl)thiazole-2(3H)-thione as a Photochemical Hydroxyl-radical Source: Photochemistry and Oxidative Damage of DNA (Strand Breaks) and 2′-Deoxyguanosine (8-oxodG Formation)¶

Inhibitory Effect of Natural Coumarin Compounds, Esculetin and Esculin, on Oxidative DNA Damage and Formation of Aberrant Crypt Foci and Tumors Induced by 1,2-Dimethylhydrazine in Rat Colons

The effects of esculetin (6,7-dihydroxycoumarin) and its 6-glycoside, esculin, on 8-oxo-2'-deoxyguanosine (8-oxodG) formation and carcinogenesis induced by a chemical carcinogen, 1,2-dimethylhydrazine (DMH), were examined in the colons of male Fischer 344 rats. Animals were given water containing esculetin or esculin for 7 d before subcutaneous injection of DMH (20 mg/kg body wt), killed 24 h after DMH treatment, and the levels of thiobarbituric acid reactive substances (TBARS) and 8-oxodG in the colons were determined. Both esculetin and esculin suppressed significantly the DMH-induced increases in 8-oxodG and TBARS in rat colon mucosa. We further investigated the modifying effect of esculin intake on the development of DMH-induced colonic aberrant crypt foci (ACF). Animals were given DMH once a week for 4 weeks to induce ACF. They then received water containing esculin ad libitum for 5 weeks (initiation phase) or 11 weeks after DMH treatment (post-initiation phase). Animals in the positive control group received tap water throughout the experiment. At the end of the experiment (16 weeks), the ingestion of esculin during the initiation phase significantly reduced the incidence of gross tumors, the number of ACF per rat and the mean number of AC per focus, while the esculin treatment during the post-initiation phase significantly decreased only the number of ACF per rat. These results suggest that esculin intake has an inhibitory effect on DMH-induced oxidative DNA damage and carcinogenesis in rat colons.

Oxidative DNA damage induced by hair dye components ortho-phenylenediamines and the enhancement by superoxide dismutase

There is an association between occupational exposure to hair dyes and incidence of cancers. Permanent oxidant hair dyes are consisted of many chemical components including ortho-phenylenediamines. To clarify the mechanism of carcinogenesis by hair dyes, we examined DNA damage induced by mutagenic ortho-phenylenediamine ( o-PD) and its derivatives, 4-chloro- ortho-phenylenediamine (Cl-PD) and 4-nitro- ortho-phenylenediamine (NO 2-PD), using 32P-labeled DNA fragments obtained from the human p16 and the p53 tumor suppressor gene. We also measured the content of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage, in calf thymus DNA with an electrochemical detector coupled to a high performance liquid chromatograph. Carcinogenic o-PD and Cl-PD caused Cu(II)-mediated DNA damage, including 8-oxodG formation, and antioxidant enzyme superoxide dismutase (SOD) enhanced DNA damage. o-PD and Cl-PD caused piperidine-labile and formamidopyrimidine-DNA glycosylase-sensitive lesions at cytosine and guanine residues respectively in the 5′-ACG-3′ sequence, complementary to codon 273, a well-known hotspot of the human p53 tumor suppressor gene. UV–vis spectroscopic studies showed that the spectral change of o-PD and Cl-PD required Cu(II), and addition of SOD enhanced it. This suggested that SOD enhanced the rate of Cu(II)-mediated autoxidation of o-PD and Cl-PD, leading to enhancement of DNA damage. On the other hand, mutagenic but non-carcinogenic NO 2-PD induced no DNA damage. These results suggest that carcinogenicity of ortho-phenylenediamines is associated with ability to cause oxidative DNA damage rather than bacterial mutagenicity.

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-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<0.05). Western blotting and immunohistochemistry revealed that the expression of NF-kappaB 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.

Lipids and DNA oxidation in Staphylococcus aureus as a consequence of oxidative stress generated by ciprofloxacin

Ciprofloxacin induced an increment of reactive oxygen species in sensitive strains of Staphylococcus aureus leading to oxidative stress detected by chemiluminescence while resistant strains did not suffer such stress. Oxidation of lipids was performed by employing thiobarbituric acid reaction to detect the formation of the amplified intermediate between reactive species oxygen and cytoplasmic macromolecules, namely malondialdehyde (MDA). The sensitive strain presented higher peroxidation of lipids than the resistant strain. The oxidative consequence for DNA was investigated by means of bacteria incubation with ciprofloxacin and posterior extraction of DNA, which was studied by high performance liquid chromatography (HPLC). Sensitive S. aureus ATCC 29213 showed an increase of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) respect controls without antibiotic; there was evident increase of the ratio between 8-oxodG and deoxyguanosine (dG) as a consequence of oxidation of dG to 8-oxodG considered the major DNA marker of oxidative stress. The resistant strain showed low oxidation of DNA and the analysis of 8-oxodG/dG ratio indicated lesser formation of 8-oxodG than S. aureus ATCC 29213.

Etiology of bromate-induced cancer and possible modes of action-studies in Japan

Renal cell tumors were significantly increased in male and female rats given potassium bromate at 250 and 500 mg/L in drinking water. In at least one other study renal cell tumors were produced in male rats at 125 mg/L. Among male mice given 750 mg/L of potassium bromate, there were no significant differences in renal cell tumors between treated and control groups after 88 weeks on test. In oxidative DNA damage tests 8-oxodeoxyguanosine (8-oxodG also referred to as 8-OH-dG) was induced in DNA in the male rat kidney in 1 week, and in females after 3 weeks at 500 mg/L, and also in both male and female rats at 250 mg/L, but not at 125 mg/L. DNA adducts are considered to be an initial step in the carcinogenesis process, however, the administered doses are not always sufficient to cause mutations, possibly due to DNA repair. In the two-step rat renal carcinogenesis model using N-ethyl- N-hydroxyethylnitrosamine (EHEN) as initiator, promotion activity by potassium bromate was measured using the BrdU labeling index. The promoting activity of bromate in male rats was much greater and extended to doses as low as 60 mg/L in male rats, whereas in females the response was limited to 250 and 500 mg/L. Therefore, it was concluded that the mechanisms contributing to cancer in the male rat were more complex than in the female rat. The accumulation of α 2μ-globulin in the kidneys of male rats exposed to potassium bromate probably accounts for the greater labeling index in the male rat relative to the female rat. Accumulation of α 2μ-globulin as a result of treatment with chemicals is unique to the male rat and does contribute to carcinogenic responses. Neither humans nor female rats display this response. Nevertheless, bromate must be considered carcinogenic because of the response of the female rats. The better correlation between 8-oxodG formation and tumor response indicates that dose–response information from the female rat would be much more relevant to human risk assessment. The fact that an elevation of BrdU-LI in the kidney of the female rat is consistent with the possibility that cell proliferation observed in female rats resulted from oxidative stress and/or cytotoxic responses in the kidney. Therefore, oxidative stress is most likely the mechanism of interest for cancer risk in humans.

Mechanism of DNA damage induced by bromate differs from general types of oxidative stress

A representative reactive oxygen species (ROS), hydroxyl radical ( OH), is a highly reactive species and induces DNA backbone breakage. OH also oxidizes every DNA base. The interaction of OH with guanine leads to the generation of not only piperidine-resistant 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) but also various piperidine-labile products. On the other hand, potassium bromate (KBrO 3) induces specific formation of 8-oxodG in the presence of SH compounds, such as glutathione (GSH) and cysteine (Cys). GSH/Cys reduces KBrO 3 (BrO 3 −) to BrO 2, which abstracts one electron from guanine. The one-electron oxidation of guanine may yield cation radicals followed by the reaction with a water molecule, leading to 8-oxodG formation. Therefore, mechanism of bromate-induced oxidative DNA damage is different from general types of oxidative stress such as OH.

Radical production and DNA damage induced by carcinogenic 4-hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus

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 32P-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 H2O2 in the DNA damage. These findings suggest that H2O2 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 μM) 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 H2O2 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.

Formation of DNA Damaging Product from Light-Irradiated Nonylphenol

Nonylphenol (NP) is widely distributed in the environment as a microbial metabolite of nonylphenol polyethoxylates, which are commercially important surfactants. Although there are concerns regarding the estrogenic activity of NP and its effects on wildlife, this paper reports a new detrimental effect for NP; oxidative DNA damage. After UV or sunlight irradiation, several NP photoproducts were detected by high performance liquid chromatography (HPLC). Two HPLC fractions induced formation of 8-oxo-7,8-dihydro-2′deoxyguanosine (8-oxodG) in calf thymus DNA. Based on gas chromatography/mass spectrometry and UV/ visible spectrometry, one fraction was confirmed to contain 4-nonylcatechol. The other active compound remains unidentified. Non-irradiated NP did not exhibit any detectable 8-oxodG formation. These results suggest that NP induces oxidative DNA damage following environmental light exposure. This DNA damaging activity is a new adverse effect of NP and, in addition to estrogenic activity, should be investigated further and taken into consideration during NP risk assessment.

Carcinogenic 3-nitrobenzanthrone induces oxidative damage to isolated and cellular DNA

3-Nitrobenzanthrone (3-NBA) is an extremely potent mutagen in diesel exhaust. It is a lung carcinogen to rats, and therefore a suspected carcinogen to human. In order to clarify the mechanism of carcinogenicity of 3-NBA, we investigated oxidative DNA damage by N-hydroxy-3-aminobenzanthrone (N-OH-ABA), a metabolite of 3-NBA, using 32P-labeled DNA fragments from the human p53 tumor-suppressor gene. N-OH-ABA caused Cu(II)-mediated DNA damage, and endogenous reductant NADH dramatically enhanced this process. Catalase and a Cu(I)-specific chelator decreased DNA damage, suggesting the involvement of hydrogen peroxide (H 2O 2) and Cu(I). N-OH-ABA induced DNA damage at cytosine and guanine residues of ACG sequence complementary to codon 273, a well-known hot spot of the p53 gene. N-OH-ABA dose dependently induced 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) formation in the presence of Cu(II) and NADH. Treatment with N-OH-ABA increased amounts of 8-oxodG in HL-60 cells compared to the H 2O 2-resistant clone HP100, supporting the involvement of H 2O 2. The present study has demonstrated that the N-hydroxy metabolite of 3-NBA induces oxidative DNA damage through H 2O 2 in both a cell-free system and cultured human cells. We conclude that oxidative DNA damage may play an important role in the carcinogenic process of 3-NBA in addition to previously reported DNA adduct formation.

Procyanidin B2 has anti- and pro-oxidant effects on metal-mediated DNA damage

Procyanidin B2 (epicatechin-(4β-8)-epicatechin), which is present in grape seeds, apples, and cacao beans, has antioxidant properties. We investigated the mechanism of preventive action of procyanidin B2 against oxidative DNA damage in human cultured cells and isolated DNA. Procyanidin B2 inhibited the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in the human leukemia cell line HL-60 treated with an H 2O 2-generating system. In contrast, a high concentration of procyanidin B2 increased the formation of 8-oxodG in HL-60 cells. Experiments with calf thymus DNA also revealed that procyanidin B2 decreased 8-oxodG formation by Fe(II)/H 2O 2, whereas procyanidin B2 induced DNA damage in the presence of Cu(II), and H 2O 2 extensively enhanced it. An electron spin resonance spin trapping study utilizing 3,3,5,5-tetramethyl-1-pyrroline- N-oxide (M 4PO) demonstrated that procyanidin B2 decreased the signal of M 4PO–OH from H 2O 2 and Fe(II), whereas procyanidin B2 enhanced the signal from H 2O 2 and Cu(II). As an antioxidant mechanism, UV–visible spectroscopy showed that procyanidin B2 chelated Fe(II) at equivalent concentrations. As a pro-oxidant property, we examined DNA damage induced by procyanidin B2, using 32P-labeled DNA fragments obtained from genes relevant to human cancer. Our results raise the possibility that procyanidin B2 exerts both antioxidant and pro-oxidant properties by interacting with H 2O 2 and metal ions.

Inhibitory effect of green tea extract on β-amyloid-induced PC12 cell death by inhibition of the activation of NF-κB and ERK/p38 MAP kinase pathway through antioxidant mechanisms

Beta-amyloid peptide (Aβ) is considered responsible for the pathogenesis of Alzheimer's disease (AD). Several lines of evidence support that Aβ-induced cytotoxicity is mediated through the generation of reactive oxygen species (ROS). Thus, agents that scavenge ROS level may usefully impede the development or progress of AD. Green tea extract has been known to have such antioxidant properties. Our previous studies demonstrate that green tea extract protected ischemia/reperfusion-induced brain cell death by scavenging oxidative damages of macromolecules. In this study, we investigated the effects of green tea extract on Aβ-induced oxidative cell death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with Aβ 25–35 (10–50 μM) showed intracellular ROS elevation, the formation of 8-oxodG (an oxidized form of DNA), and underwent apoptotic cell death in a dose-dependent manner. Aβ 25–35 treatment upregulated pro-apoptotic p53 at the gene level, and Bax and caspase-3 at the protein level, but downregulated anti-apoptotic Bcl-2 protein. Interestingly, co-treated green tea extract (10–50 μg/ml) dose-dependently attenuated Aβ 25–35 (50 μM)-induced cell death, intracellular ROS levels, and 8-oxodG formation, in addition to p53, Bax, and caspase-3 expression, but upregulated Bcl-2. Furthermore, green tea extract prevented the Aβ 25–35-induced activations of the NF-κB and ERK and p38 MAP kinase pathways. Our study suggests that green tea extract may usefully prevent or retard the development and progression of AD.

Effect of sesaminol glucosides on β-amyloid-induced PC12 cell death through antioxidant mechanisms

Several lines of evidence support that beta-amyloid (Aβ)-induced neurotoxicity is mediated through the generation of reactive oxygen species (ROS) and elevation of intracellular calcium. In this study, we have investigated protective effects of sesaminol glucosides on Aβ-induced oxidative cell death in cultured rat pheochromocytoma (PC12) cells. Sesaminol glucoside (50–250 μg/ml) decreased Aβ 25–35-induced ROS generation, formation of 8-oxodG, a form of oxidative DNA and elevation of intracellular calcium level concomitant with prevention of apoptotic cell death dose dependently. Sesaminol glucoside (50–250 μg/ml) also effectively decreased Aβ 1–42 and ADDL form of Aβ 1–42 as well as the combination of H 2O 2 with FeSO 4-induced cell damages. In mechanistic study, sesaminol glucosides attenuated Aβ 25–35-induced activation of redox transcription factor nuclear factor-κB (NF-κB) through inhibition of p50 translocation and IκB phosphorylation, and blocked NF-κB-dependent luciferase activity in addition to the inhibitory effect on Aβ 25–35-induced activation of ERK kinase signal pathway. Consistent with the inhibitory effect on Aβ 25–35-induced stress-induced cell death, sesaminol glucosides decreased expression of pro-apoptotic gene p53, and Bax and caspase-3, but enhanced expression of anti-apoptotic Bcl-2. Moreover, the protective effects of sesaminol glucoside on Aβ 25–35-induced ROS generation, NF-κB activation and cell death were further enhanced with glutathione. This study therefore suggests that sesaminol glucosides have protective effect on Aβ-induced neuronal cell death, and its effect may be through antioxidative property.