Shosuke Kawanishi

Oxidative DNA damage induced by simultaneous generation of nitric oxide and superoxide.

Incubation of calf thymus DNA with 3‐morpholinosydnonimine (SIN‐1), which simultaneously generates nitric oxide (NO) and superoxide (O2 −), induced a significant increase of 8‐hydroxydeoxyguanosine (8‐OH‐dG). Peroxynitrite also increased 8‐OH‐dG in calf thymus DNA. Addition of free hydroxyl radical (•OH) scavengers inhibited the increase of 8‐OH‐dG by SIN‐1 or peroxynitrite. Incubation of 32P‐labeled DNA fragment with SIN‐1 or peroxynitrite caused DNA cleavage at every nucleotide with a little dominance at guanine residues. The results suggest that NO reacts with O2 − to form peroxynitrite and the peroxynitrite induces oxidative DNA damage through an active intermediate of which reactivity is similar to •OH.

Oxidative and nitrative DNA damage in animals and patients with inflammatory diseases in relation to inflammation-related carcinogenesis.

Abstract Infection and chronic inflammation are proposed to contribute to carcinogenesis through inflammation-related mechanisms. Infection with hepatitis C virus, Helicobacter pylori and the liver fluke, Opisthorchis viverrini (OV), are important risk factors for hepatocellular carcinoma (HCC), gastric cancer and cholangiocarcinoma, respectively. Inflammatory bowel diseases (IBDs) and oral diseases, such as oral lichen planus (OLP) and leukoplakia, are associated with colon carcinogenesis and oral squamous cell carcinoma (OSCC), respectively. We performed a double immunofluorescence labeling study and found that nitrative and oxidative DNA lesion products, 8-nitroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), were formed and inducible nitric oxide synthase (iNOS) was expressed in epithelial cells and inflammatory cells at the site of carcinogenesis in humans and animal models. Antibacterial, antiviral and antiparasitic drugs dramatically diminished the formation of these DNA lesion markers and iNOS expression. These results suggest that oxidative and nitrative DNA damage occurs at the sites of carcinogenesis, regardless of etiology. Therefore, it is considered that excessive amounts of reactive nitrogen species produced via iNOS during chronic inflammation may play a key role in carcinogenesis by causing DNA damage. On the basis of our results, we propose that 8-nitroguanine is a promising biomarker to evaluate the potential risk of inflammation-mediated carcinogenesis.

Mechanism of Telomere Shortening by Oxidative Stress

Abstract: We investigated whether oxidative stress, which contributes to aging, accelerates the telomere shortening in human cultured cells. The terminal restriction fragment (TRF) from WI‐38 fibroblasts irradiated with UVA (365‐nm light) decreased with increasing of the irradiation dose. Furthermore, UVA irradiation dose‐dependently increased the formation of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) in both WI‐38 fibroblasts and HL‐60 cells. In order to clarify the mechanism of the acceleration of telomere shortening, we investigated site‐specific DNA damage induced by UVA irradiation in the presence of endogenous photosensitizers using 32P 5′ end‐labeled DNA fragments containing telomeric oligonucleotide (TTAGGG)4. UVA irradiation with riboflavin induced 8‐oxodG formation in the DNA fragments containing telomeric sequence, and Fpg protein treatment led to chain cleavages at the central guanine of 5′‐GGG‐3′ in telomere sequence. Human 8‐oxodG‐DNA glycosylase introduces a chain break in a double‐stranded oligonucleotide specifically at an 8‐oxodG residue. The amount of 8‐oxodG formation in DNA fragment containing telomere sequence [5′‐CGC(TTAGGG)7CGC‐3′] was approximately five times more than that in the DNA fragment containing nontelomere sequence [5′‐CGC(TGTGAG)7CGC‐3′]. Furthermore, H2O2 plus Cu(II) caused DNA damage, including 8‐oxodG formation, specifically at the GGG sequence in the telomere sequence (5′‐TTAGGG‐3′). It is concluded that the formation of 8‐oxodG at the GGG triplet in telomere sequence induced by oxidative stress could participate in acceleration of telomere shortening.

Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening.

Telomere shortening during human aging has been reported to be accelerated by oxidative stress. We investigated the mechanism of telomere shortening by oxidative stress. H2O2 plus Cu(II) caused predominant DNA damage at the 5′ site of 5′‐GGG‐3′ in the telomere sequence. Furthermore, H2O2 plus Cu(II) induced 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) formation in telomere sequences more efficiently than that in non‐telomere sequences. NO plus O2 − efficiently caused base alteration at the 5′ site of 5′‐GGG‐3′ in the telomere sequence. It is concluded that the site‐specific DNA damage at the GGG sequence by oxidative stress may play an important role in increasing the rate of telomere shortening with aging.

The role of metals in site-specific DNA damage with reference to carcinogenesis

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.

Photo-irradiated Titanium Dioxide Catalyzes Site Specific DNA Damage via Generation of Hydrogen Peroxide

Titanium dioxide (TiO2) is a potential photosensitizer for photodynamic therapy. In this study, the mechanism of DNA damage catalyzed by photo-irradiated TiO2 was examined using [32P]-5′-end-labeled DNA fragments obtained from human genes. Photo-irradiated TiO2 (anatase and rutile) caused DNA cleavage frequently at the guanine residue in the presence of Cu(II) after E. coli formamidopyrimidine-DNA glycosylase treatment, and the thymine residue was also cleaved after piperidine treatment. Catalase, SOD and bathocuproine, a chelator of Cu(I), inhibited the DNA damage, suggesting the involvement of hydrogen peroxide, superoxide and Cu(I). The photocatalytic generation of Cu(I) from Cu(II) was decreased by the addition of SOD. These findings suggest that the inhibitory effect of SOD on DNA damage is due to the inhibition of the reduction of Cu(II) by superoxide. We also measured the formation of 8-oxo-7,8-dihydro-2′ -deoxyguanosine, an indicator of oxidative DNA damage, and showed that anatase is more active than rutile. On the other hand, high concentration of anatase caused DNA damage in the absence of Cu(II). Typical free hydroxyl radical scavengers, such as ethanol, mannnitol, sodium formate and DMSO, inhibited the copper-independent DNA photodamage by anatase. In conclusion, photo-irradiated TiO2 particles catalyze the copper-mediated site-specific DNA damage via the formation of hydrogen peroxide rather than that of a free hydroxyl radical. This DNA-damaging mechanism may participate in the phototoxicity of TiO2.

Oxidative DNA damage by vitamin A and its derivative via superoxide generation.

Recent intervention studies revealed that β-carotene supplement to smokers resulted in a higher incidence of lung cancer. However, the causal mechanisms remain to be clarified. We reported here that vitamin A (retinol) and its derivative (retinal) caused cellular DNA cleavage detected by pulsed field gel electrophoresis. Retinol and retinal significantly induced 8-oxo-7,8-dihydro-2′-deoxyguanosine formation in HL-60 cells but not in H2O2-resistant HP100 cells, suggesting the involvement of H2O2 in cellular DNA damage. Experiments using 32P-labeled isolated DNA demonstrated that retinol and retinal caused Cu(II)-mediated DNA damage, which was inhibited by catalase. UV-visible spectroscopic and electron spin resonance-trapping studies revealed the generation of superoxide and carbon-centered radicals, respectively. The superoxide generation during autoxidation of retinoids was significantly correlated with the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine, although the yield of carbon-centered radicals was not necessarily related to the intensity of DNA damage. These findings suggest that superoxide generated by autoxidation of retinoids was dismutated to H2O2, which was responsible for DNA damage in the presence of endogenous metals. Retinol and retinal have prooxidant abilities, which might lead to carcinogenesis of the supplements of β-carotene.

Mechanism of oxidative DNA damage induced by quercetin in the presence of Cu(II)

Quercetin, one of flavonoids, has been reported to be carcinogenic. There have been no report concerning carcinogenicity of kaempferol and luteolin which have structure similar to quercetin. DNA damage was examined by using DNA fragments obtained from the human p53 tumor suppressor gene. Quercetin induced extensive DNA damage via reacting with Cu(II), but kaempferol and luteolin induced little DNA damage even in the presence of Cu(II). Excessive quercetin inhibited copper-dependent DNA damage induced by quercetin. Bathocuproine, a Cu(I)-specific chelator, catalase and methional inhibited the DNA damage by quercetin, whereas free hydroxyl radical scavengers did not. Site specificity of the DNA damage was thymine and cytosine residues. The site specificity and the inhibitory effects suggested that DNA-copper-oxygen complex rather than free hydroxyl radical induced the DNA damage. Formation of 8-oxodG by quercetin increased extensively in the presence of Cu(II), whereas 8-oxodG formation by kaempferol or luteolin increased only slightly. This study suggests a good relationship between carcinogenicity and oxidative DNA damage of three flavonoids. The mechanism of DNA damage by quercetin was discussed in relation to the safety in cancer chemoprevention by flavonoids.

H2O2 Accelerates Cellular Senescence by Accumulation of Acetylated p53 via Decrease in the Function of SIRT1 by NAD+ Depletion

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 H₂O₂ and examined DNA cleavage, depletion of intracellular NAD⁺, expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated β-galactosidase (SA-β-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H₂O₂, though no cell death was observed. NAD⁺ levels in TIG-3 cells treated with H₂O₂ 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 H₂O₂ 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 H₂O₂ induced cell cycle arrest at 24h and increased SA-β-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.

Superoxide formation and DNA damage induced by a fragrant furanone in the presence of copper(II)

2,5-Dimethyl-4-hydroxy-3(2H)-furanone (2,5-DMHF), a caramel-like fragrant compound found in may processed foodstuff, has been reported to be mutagenic. 4,5-Dimethyl-3-hydroxy-2(5H)-furanone (4,5-DMHF), which is a similar characteristic fragrant compound, has no report concerning its mutagenicity. DNA damage by 2,5-DMHF and 4,5-DMHF was investigated by using DNA fragments obtained from the p53 tumor suppressor gene. 2,5-DMHF induced DNA damage extensively in the presence of Cu(II), but only slightly in the presence of Fe(III). 4,5-DMHF did not cause metal-dependent DNA damage. Bathocuproine, a Cu(I)-specific chelator, and catalase inhibited DNA damage induced by 2,5-DMHF plus Cu(II), whereas free hydroxyl radical scavengers did not. The order of DNA cleavage sites was thymine, cytosine > guanine residues. The site-specific DNA damage and effects of scavengers show that DNA-copper-oxygen complex rather than free .OH are involved in the DNA damage. Formation of 8-oxodeoxyguanosine (8-oxodG) by 2,5-DMHF increased with its concentration in the presence of Cu(II), whereas 8-oxodG formation increased only slightly in the presence of Fe(III). Degradation of 2,5-DMHF was efficiently accelerated by Cu(II), but only slightly accelerated by Fe(III). The degradation of 4,5-DMHF was little even in the presence of metal ions. Examination using cytochrome c suggest that superoxide was generated from 2,5-DMHF. Stoichiometric study of Cu(II) reduction revealed that autoxidation of 2,5-DMHF could offer 4-electron reduction. These results suggest that, at least in vitro and in an acellular system, 2,5-DMHF generates superoxide and subsequently hydrogen peroxide to induce metal-dependent DNA damage.