Oxidative damage to DNA induced by areca nut extract

In Taiwan, people chew bete; quid which contains tender areca nut with husk. In other countries, people prefer ripe and dried areca nut without husk. In this study, we compared the reactive oxygen species-induced oxidative DNA damage in isolated DNA and CHO-K1 cells between treatments with tender areca nut extract (ANE) and ripe ANE. Incubation of these two ANE preparations with isolated DNA generated 8-hydroxy-2′-deoxyguanosine (8-OH-dG) in an alkaline environment in a dose-dependent manner. Ripe ANE generated higher levels of 8-OH-dG compared to tender ANE. The addition of iron(II) (100 μM) resulted in 1.4- and 3.1-fold increases of 8-OH-dG when incubated with 1 mg/ml each of tender and ripe ANE. In testing the effect of ANE to cellular DNA, CHO-K1 cells were used for its documented sensitivity to reactive oxygen species. In CHO-K1 cells, ripe ANE was more cytotoxic than tender ANE following an 18-h incubation. The cytotoxicity to CHO-K1 cells was positively correlated with the formation of 8-OH-dG following tender (r = 0.97) and ripe (r = 0.91) ANE treatment. Addition of the iron chelating agent o-phenanthroline (10 and 20 μM) to cells prior to ripe ANE exposure significantly increased (p < 0.05) the survival of CHO-K1 cells. In addition, ripe ANE induced dichlorofluorescein-mediated fluorescence which indicated the formation of hydrogen peroxide in CHO-K1 cells. In conclusion, this study demonstrated that ANE-induced oxidative damage to isolated and cellular DNA which may result from the generation of hydrogen peroxide, and iron may serve as a catalyst in this process. Furthermore, ripe ANE generated higher oxidative DNA damage levels compared to tender ANE.     

Oxidative DNA damage induced by a hydroperoxide derivative of cyclophosphamide

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.     

The paradoxical nature of DNA damage and cell death induced by 125I decay.

Chinese hamster ovary cells were synchronized at the G1/S-phase boundary of the cell cycle and pulse-labeled for 10 min with 125I-iododeoxyuridine 30 min after entering the S phase. Cell samples were harvested for freezing and 125I-decay accumulation at intervals ranging from 15 to 480 min after termination of labeling. The survival data showed a marked shift from cell killing characteristic of low-LET radiation to that more characteristic of killing by high-LET radiation with increasing intervals between DNA pulse-labeling and decay accumulation. Cells harvested and frozen within 1 h after pulse-labeling yielded a low-LET radiation survival response with a pronounced shoulder and a large D0 of up to 0.9 Gy. With longer chase periods the shoulder and the D0 decreased progressively, and cells harvested 5 h after pulse-labeling or later exhibited a high-LET survival response (D0: 0.13 Gy). Two interpretations for these findings are discussed. (1) If DNA is the sole target for radiation death, the results indicate that DNA maturation increases radiation damage to DNA or reduces damage repair. (2) If radiation cell death involves damage to higher-order structures in the cell nucleus, the findings suggest that newly replicated DNA is not attached to these structures during the initial low-LET period, but 125I starts to induce high-LET radiation effects as labeled DNA segments become associated with the target structure(s). On balance, or data favor the latter interpretation.     

Oxidative DNA damage induced by aminoacetone, an amino acid metabolite

We investigated DNA damage induced by aminoacetone, a metabolite of threonine and glycine. Pulsed-field gel electrophoresis revealed that aminoacetone caused cellular DNA cleavage. Aminoacetone increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in human cultured cells in a dose-dependent manner. The formation of 8-oxodG in calf thymus DNA increased due to aminoacetone only in the presence of Cu(II). DNA ladder formation was observed at higher concentrations of aminoacetone than those causing DNA cleavage. Flow cytometry showed that aminoacetone enhanced the generation of hydrogen peroxide (H2O2) in cultured cells. Aminoacetone caused damage to 32P-5'-end-labeled DNA fragments, obtained from the human c-Ha-ras-1 and p53 genes, at cytosine and thymine residues in the presence of Cu(II). Catalase and bathocuproine inhibited DNA damage, suggesting that H2O2 and Cu(I) were involved. Analysis of the products generated from aminoacetone revealed that aminoacetone underwent Cu(II)-mediated autoxidation in two different pathways: the major pathway in which methylglyoxal and NH+4 are generated and the minor pathway in which 2,5-dimethylpyrazine is formed through condensation of two molecules of aminoacetone. These findings suggest that H2O2 generated by the autoxidation of aminoacetone reacts with Cu(I) to form reactive species capable of causing oxidative DNA damage.     

Oxidative DNA damage induced by nitrotyrosine, a biomarker of inflammation

Inflammation has been postulated as a risk factor for several cancers. 3-Nitrotyrosine is a biochemical marker for inflammation. We investigated the ability of nitrotyrosine and nitrotyrosine-containing peptides (nitroY-peptide) to induce DNA damage by the experiments using 32P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and an HPLC-electrochemical detector. Nitrotyrosine and nitroY-peptide caused Cu(II)-dependent DNA damage in the presence of P450 reductase, which is considered to yield nitroreduction. Catalase inhibited DNA damage, suggesting the involvement of H2O2. Nitrotyrosine and nitroY-peptide increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, an indicator of oxidative DNA damage. Nitrotyrosine-containing peptides of histone induced 8-oxodG formation more efficiently than free nitrotyrosine. We propose the possibility that nitrotyrosine-induced H2O2 formation and DNA damage contribute to inflammation-associated carcinogenesis.     

Proteasome inhibition prevents cell death induced by the chemotherapeutic agent cisplatin downstream of DNA damage

Cisplatin is a highly effective chemotherapeutic drug acting as a DNA-damaging agent that induces apoptosis of rapidly proliferating cells. Unfortunately, cellular resistance still occurs. Mutations in p53 in a large fraction of tumor cells contribute to defects in apoptotic pathways and drug resistance. To uncover new strategies to eliminate tumors through a p53-independent pathway, we established a simplified model devoid of p53 to study cisplatin-induced regulated cell death, using the yeast Saccharomyces cerevisiae. We previously showed that cisplatin induces an active form of cell death accompanied by DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction. We further demonstrated that proteasome inhibition, either with MG132 or genetically, increased resistance to cisplatin. In this study, we sought to determine how proteasome inhibition is important for cisplatin resistance by analyzing how it affects several phenotypes associated with the DNA damage response. We found MG132 does not seem to affect the activation of the DNA damage response or increase damage tolerance. Moreover, central modulators of the DNA damage response are not required for cisplatin resistance imparted by MG132. These results suggest the proteasome is involved in modulation of cisplatin toxicity downstream of DNA damage. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Elucidation of this mechanism will therefore aid in the development of new strategies to increase the efficacy of chemotherapy.     

Oxidative damage of DNA induced by the cytochrome C and hydrogen peroxide system

To elaborate the peroxidase activity of cytochrome c in the generation of free radicals from H2O2, the mechanism of DNA cleavage mediated by the cytochrome c/H2O2 system was investigated. When plasmid DNA was incubated with cytochrome c and H2O2, the cleavage of DNA was proportional to the cytochrome c and H2O2 concentrations.Radical scavengers, such as azide, mannitol, and ethanol, significantly inhibited the cytochrome c/H2O2 system-mediated DNA cleavage. These results indicated that free radicals might participate in the DNA cleavage by the cytochrome c and H2O2 system. Incubation of cytochrome c with H2O2 resulted in a time-dependent release of iron ions from the cytochrome c molecule. During the incubation of deoxyribose with cytochrome c and H2O2, the damage to deoxyribose increased in a time-dependent manner, suggesting that the released iron ions may participate in a Fenton-like reaction to produce dOH radicals that may cause the DNA cleavage. Evidence that the iron-specific chelator, desferoxamine (DFX), prevented the DNA cleavage induced by the cytochrome c/H2O2 system supports this mechanism. Thus we suggest that DNA cleavage is mediated via the generation of dOH by a combination of the peroxidase reaction of cytochrome c and the Fenton-like reaction of free iron ions released from oxidatively damaged cytochrome c in the cytochrome c/H2O2 system.     

Oxidative DNA damage induced by metabolites of chloramphenicol,an antibiotic drug

Abstract

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 ³²P-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.     

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 (32)P-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.     

Oxidative DNA damage induced by toluene is involved in its male reproductive toxicity

Toluene is widely used as an organic solvent in various industries and commercial products. Recent investigations have shown that toluene may induce male reproductive dysfunctions and carcinogenicity. To clarify whether the toxicity results from the interference of endocrine systems or direct damage to reproductive organs, we examined the effects of toluene on the male reproductive system in rats, comparing to those of diethylstilbestrol (DES), a potent synthetic estrogen. Toluene (50, 500 mg/kg) or DES (2 mg/kg) injected subcutaneously to male Sprague-Dawley rats once a day for 10 days decreased the epididymal sperm counts and the serum concentrations of testosterone. The mRNA level for gonadotropin-releasing hormone receptor in the pituitary was decreased by DES, but not by toluene. On the contrary, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in testes, the biological marker for oxidative DNA damage, was increased by toluene but not by DES. These results suggest that toluene induces reproductive toxicity via direct oxidative damage of spermatozoa, whereas DES affects endocrine systems via the hypothalamo-pituitary-gonadal axis. Morphological findings supported the idea. To determine the mechanism of 8-oxodG formation in vivo , we examined DNA damage induced by toluene metabolic products in vitro . Minor toluene metabolites, methylhydroquinone and methylcatechols, induced oxidative DNA damage, and the methylcatechols induced NADH-mediated 8-oxodG formation more efficiently than methylhydroquinone did. We propose that oxidative DNA damage in the testis plays a role in reproductive toxicity induced by toluene.     

Oxidative DNA damage in human peripheral leukocytes induced by massive aerobic exercise.

Reactive oxygen species produced during vigorous exercise may permeate into cell nuclei and induce oxidative DNA damage, but the supporting evidence is still lacking. By using a 42 km marathon race as a model of massive aerobic exercise, we demonstrated a significant degree of unrepaired DNA base oxidation in peripheral immunocompetent cells, despite a concurrent increase in the urinary excretion of 8-hydroxy-2'-deoxyguanosine. Single cell gel electrophoresis with the incorporation of lesion-specific endonucleases further revealed that oxidized pyrimidines (endonuclease III-sensitive sites) contributed to most of the postexercise nucleotide oxidation. The oxidative DNA damage correlated significantly with plasma levels of creatinine kinase and lipid peroxidation metabolites, and lasted for more than 1 week following the race. This phenomenon may be one of the mechanisms behind the immune dysfunctions after exhaustive exercise.     

Oxidative DNA base damage induced by singlet oxygen and photosensitization: recognition by repair endonucleases and mutagenicity

We have analyzed the recognition by various repair endonucleases of DNA base modifications induced by three oxidants, viz. [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), a photochemical source of tert-butoxyl radicals, disodium salt of 1,4-etheno-2,3-benzodioxin-1,4-dipropanoic acid (NDPO(2)), a chemical source of singlet oxygen, and riboflavin, a type-I photosensitizer. The base modifications induced by BCBT, which were previously shown to be mostly 7,8-dihydro-8-oxoguanine (8-oxoGua) residues, were recognized by Fpg and Ogg1 proteins, but not by endonuclease IIII, Ntg1 and Ntg2 proteins. In the case of singlet oxygen induced damage, 8-oxoGua accounted for only 35% of the base modifications recognized by Fpg protein. The remaining Fpg-sensitive modifications were not recognized by Ogg1 protein and relatively poor by endonuclease III, but they were relatively good substrates of Ntg1 and Ntg2. In the case of the damage induced by photoexcited riboflavin, the fraction of Fpg-sensitive base modifications identified as 8-oxoGua was only 23%. In contrast to the damage induced by singlet oxygen, the remaining lesions were not only recognized by Ntg1 and Ntg2 proteins and (relatively poor) by endonuclease III, but also by Ogg1 protein. The analysis of the mutations observed after transfection of modified plasmid pSV2gpt into Escherichia coli revealed that all agents induced near exclusively GC-->TA and GC-->CG transversions, the numbers of which were correlated with the numbers of 8-oxoGua residues and Ntg-sensitive modifications, respectively. In conclusion, both singlet oxygen and the type-I photosensitizer riboflavin induce predominantly oxidative guanine modifications other than 8-oxoGua, which most probably give rise to GC-->CG transversions and in which eukaryotic cells are substrates of Ntg1 and Ntg2 proteins.     

Oxidative DNA damage induced by a metabolite of carcinogenic o-anisidine: enhancement of DNA damage and alteration in its sequence specificity by superoxide dismutase

The mechanism of DNA damage by a metabolite of the carcinogen o-anisidine in the presence of metals was investigated by the DNA sequencing technique using 32P-labeled human DNA fragments. The o-anisidine metabolite, o-aminophenol, caused DNA damage in the presence of Cu(II). The DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H2O2 and Cu(I). The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by o-aminophenol increased in the presence of Cu(II). We conclude that Cu(II)-mediated oxidative DNA damage by this o-anisidine metabolite seems to be relevant for the expression of the carcinogenicity of o-anisidine. o-Aminophenol plus Cu(II) caused preferential DNA damage at the 5'-site guanine of GG and GGG sequences. When CuZn-SOD or Mn-SOD was added, the DNA damage was enhanced and its predominant cleavage sites were changed into thymine and cytosine residues. We consider that SOD may increase the frequency of mutations due to DNA damage induced by o-aminophenol and thus increase its carcinogenic potential.     

Hydrogen peroxide induces topoisomerase I-mediated DNA damage and cell death

Reactive oxygen species modify DNA, generating various DNA lesions including modified bases such as 8-oxoguanine (8-oxoG). These base-modified DNA lesions have been shown to trap DNA topoisomerase I (TOP1) into covalent cleavage complexes. In this study, we have investigated the role of TOP1 in hydrogen peroxide toxicity. We showed that ectopic expression of TOP1 in Saccharomyces cerevisiae conferred sensitivity to hydrogen peroxide, and this sensitivity was dependent on RAD9 checkpoint function. Moreover, in the mammalian cell culture system, hydrogen peroxide-induced growth inhibition and apoptosis were shown to be partly TOP1-dependent as evidenced by a specific increase in resistance to hydrogen peroxide in TOP1-deficient P388/CPT45 murine leukemia cells as compared with their TOP1-proficient parental cell line P388. In addition, hydrogen peroxide was shown to induce TOP1-DNA cross-links. These results support a model in which hydrogen peroxide promotes the trapping of TOP1 on oxidative DNA lesions to form TOP1-DNA cleavage complexes that contribute to hydrogen peroxide toxicity.     

Oxidative stress-induced DNA damage by particulate air pollution

Exposure to ambient air particulate matter (PM) is associated with pulmonary and cardiovascular diseases and cancer. The mechanisms of PM-induced health effects are believed to involve inflammation and oxidative stress. The oxidative stress mediated by PM may arise from direct generation of reactive oxygen species from the surface of particles, soluble compounds such as transition metals or organic compounds, altered function of mitochondria or NADPH-oxidase, and activation of inflammatory cells capable of generating ROS and reactive nitrogen species. Resulting oxidative DNA damage may be implicated in cancer risk and may serve as marker for oxidative stress relevant for other ailments caused by particulate air pollution. There is overwhelming evidence from animal experimental models, cell culture experiments, and cell free systems that exposure to diesel exhaust and diesel exhaust particles causes oxidative DNA damage. Similarly, various preparations of ambient air PM induce oxidative DNA damage in in vitro systems, whereas in vivo studies are scarce. Studies with various model/surrogate particle preparations, such as carbon black, suggest that the surface area is the most important determinant of effect for ultrafine particles (diameter less than 100 nm), whereas chemical composition may be more important for larger particles. The knowledge concerning mechanisms of action of PM has prompted the use of markers of oxidative stress and DNA damage for human biomonitoring in relation to ambient air. By means of personal monitoring and biomarkers a few studies have attempted to characterize individual exposure, explore mechanisms and identify significant sources to size fractions of ambient air PM with respect to relevant biological effects. In these studies guanine oxidation in DNA has been correlated with exposure to PM(2.5) and ultrafine particles outdoor and indoor. Oxidative stress-induced DNA damage appears to an important mechanism of action of urban particulate air pollution. Related biomarkers and personal monitoring may be useful tools for risk characterization.     

Cell cycle activation linked to neuronal cell death initiated by DNA damage

Increasing evidence indicates that neurodegeneration involves the activation of the cell cycle machinery in postmitotic neurons. However, the purpose of these cell cycle-associated events in neuronal apoptosis remains unknown. Here we tested the hypothesis that cell cycle activation is a critical component of the DNA damage response in postmitotic neurons. Different genotoxic compounds (etoposide, methotrexate, and homocysteine) induced apoptosis accompanied by cell cycle reentry of terminally differentiated cortical neurons. In contrast, apoptosis initiated by stimuli that do not target DNA (staurosporine and colchicine) did not initiate cell cycle activation. Suppression of the function of ataxia telangiectasia mutated (ATM), a proximal component of DNA damage-induced cell cycle checkpoint pathways, attenuated both apoptosis and cell cycle reentry triggered by DNA damage but did not change the fate of neurons exposed to staurosporine and colchicine. Our data suggest that cell cycle activation is a critical element of the DNA damage response of postmitotic neurons leading to apoptosis.