抗癌药顺铂对小鼠脑和肾组织DNA氧化损伤的检测分析

目的:探讨抗癌药顺铂(cisplatin)对小鼠脑和肾组织DNA的氧化损伤。方法:采用SABC法进行脑和肾组织8-OHdG的免疫组化染色。结果:顺铂给药组小鼠的大脑皮质和海马的毛细血管内皮细胞及肾小球毛细血管内皮细胞呈现8-OHdG免疫组化阳性反应,对照组小鼠在上述区域未见阳性反应细胞。结论:顺铂可引起大脑皮质和海马的毛细血管内皮细胞及肾小球毛细血管内皮细胞DNA的氧化损伤。     

Different effects of genistein and resveratrol on oxidative DNA damage in vitro

Previous studies have demonstrated that phenolic compounds, including genistein (4′,5,7-trihydroxyisoflavone) and resveratrol (3,4′,5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in φX-174 plasmid DNA. H₂O₂/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H₂O₂/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H₂O₂ suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H₂O₂/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H₂O₂ were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

Hesperidin ameliorates cisplatin induced hepatotoxicity and attenuates oxidative damage,cell apoptosis,and inflammation in rats

Cisplatin is one of the most widely used chemotherapeutic anti-cancer drugs that is associated with multiple systemic toxicities limiting its use. The present study aimed to evaluate the hepato-protective effect of hesperidin against cisplatin-induced toxicity. Thirty-two adult male albino rats were equally split into four groups, the first group served as control received normal saline, the second group (CIS) received a single intraperitoneal dose of cisplatin (7.5 mg/kg bw) on the 22nd day of the experiment, the third group (HES) treated once daily with hesperidin (200 mg/kg bw, orally) for 21 days, and the last group (HES + CIS) pretreated once daily with hesperidin followed by a single intraperitoneal dose of cisplatin. Twenty-four hours later, samples were collected for further investigations. CIS-intoxication resulted in a significant decrease in the erythrogram along with thrombocytopenia leukopenia, and lymphopenia. Furthermore, CIS administration significantly elevated serum activity of liver enzymes, total, and indirect bilirubin as well serum glucose, total cholesterol, and triglycerides levels, meanwhile serum total protein, and globulin levels were significantly reduced. The hepatic MDA was markedly elevated with a concomitant decline in the hepatic antioxidant enzymes and severe alterations in the hepatic tissue architecture in CIS-intoxicated rats. Additionally, CIS-induced overexpression of hepatic Bax, caspase-3, and TNF-α, with no effect on hepatic expression of IL-10. Interestingly, HES pretreatment improved the CIS-induced hemato-biochemical, molecular and histopathological alterations. In conclusion, hesperidin hepato-protective effects against CIS might be mediated by its antioxidant, anti-inflammatory, and anti-apoptotic properties.     

Dimethylthiourea protects against mitochondrial oxidative damage induced by cisplatin in liver of rats

Cisplatin is one of the most effective chemotherapeutic agents. However, at higher doses liver injury may occur. The purpose of this study was to explore whether the hydroxyl radical scavenger dimethylthiourea (DMTU) protects against cisplatin-induced oxidative damage in vivo and to define the mitochondrial pathways involved in cytoprotection. Adult male Wistar rats (200–220 g) were divided into four groups of eight animals each. The control group was treated only with an intraperitoneal (i.p.) injection of saline solution (1 ml/100 g body weight). The DMTU group was given only DMTU (500 mg/kg body weight, i.p), followed by 125 mg/kg body weight, i.p. (twice a day) until sacrifice. The cisplatin group was given a single injection of cisplatin (10 mg/kg body weight, i.p.). The DMTU + cisplatin group was given DMTU (500 mg/kg body weight, i.p.), just before the cisplatin injection (10 mg/kg body weight, i.p.), followed by injections of DMTU (125 mg/kg body weight, i.p.) twice a day until sacrifice (72 h after the treatment). DMTU did not present any direct effect on mitochondria and substantially inhibited cisplatin-induced mitochondrial damage in liver, therefore preventing elevation of AST and ALT serum levels. DMTU protected against (a) decreased hepatic ATP levels; (b) lipid peroxidation; (c) cardiolipin oxidation; (d) sulfhydryl protein oxidation; (e) mitochondrial membrane rigidification; (f) GSH oxidation; (g) NADPH oxidation; (h) apoptosis. Results suggest that antioxidants, particularly hydroxyl radical scavengers, protect liver mitochondria against cisplatin-induced oxidative damage. Several mitochondrial changes were delineated and proposed as interesting targets for cytoprotective strategy.     

Mapping of peroxyl radical induced damage on genomic DNA

We have examined the DNA damage produced by reaction of peroxyl radicals with human fibroblast DNA. DNA damage consisted of both strand breaks and base modifications. The extent of strand breaks and base modifications induced as a function of peroxyl radical concentration was determined by quantitation of fragment size distributions using denaturing glyoxal-agarose gel electrophoresis. Both strand breaks and base modifications increased in a log linear fashion with respect to peroxyl radical concentration. Oxidative base modifications were observed to occur to a greater extent than strand breaks at every concentration measured. The sequence-specific distribution of peroxyl radical induced base damage was mapped for 803 nucleotide positions using the method of ligation mediated PCR. A total of 87% of all guanine positions in the examined sequences was found to be significantly oxidized. The order of reactivity of DNA bases toward oxidation by peroxyl radicals was found to be G > C > T. Adenine is essentially unreactive. The yield of oxidative base modifications at guanines and cytosines by peroxyl radicals depends on the exact specification of 5' and 3' flanking bases in a polarity dependent manner. Every guanine in the 5'XGC3' motif was found to be oxidized, where X is any 5' neighbor. In contrast, 5' and 3' purine flanks drastically reduced the extent of peroxyl radical G oxidation. The pattern of base modification and the influence of nearest neighbors differs substantially from that previously reported for hydrogen peroxide damage mediated by low valent transition metal ions for the identical DNA sequences.     

Determination of DNA damage induced by oxidative stress in hyperlipidemic patients

In the present paper, we report data on the genotoxic properties of hydrogen peroxide in polymorphonuclear neutrophils (PMNLs) separated from normolipidemic and type II/a hyperlipidemic patients. In all, 15 hyperlipidemic patients (11 female, 4 male, mean age 54.6±10.25 years) were involved in the study, and 7 normolipidemic patients (5 female, 2 male, mean age 53.4±8.07 years) served as controls. Using the comet assay, there was a significant difference in the degree of DNA damage between the two groups. The visual score characteristic of the degree of DNA damage was 350.97±31.31 in the hyperlipidemic group, while it was 289.5±29.49 in the control group (P<0.001). In the hyperlipidemic patients, a positive correlation was found between the degree of DNA damage and the basic oxidation of PMNLs (r=0.517), and the superoxide anion production of the cells stimulated with phorbolmiristate acetate (PMA) (r=0.326) and formyl-Met–Leu–Phe (FMLP) (r=0.525) as well. There was a negative correlation between DNA damage and HDL-associated antioxidant paraoxonase (PON) activity (r=−0.469), and the PON/HDL ratio (r=−0.631). No correlation was found between the degree of DNA damage and the plasma concentration of nitric oxide (NO) (r=0.098) and thiobarbituric acid-reactive substances (TBARS) (r=0.061) in hyperlipidemic patients. Our results show that in hyperlipidemic patients there is an increase in lymphocyte DNA damage caused by oxidative stress when compared to normolipidemic individuals as demonstrated by comet assay. Decreased antioxidant capacity in hyperlipidemic patients may play a significant role in this process.     

Effects of resveratrol and 4-hexylresorcinol on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

The protective effects of resveratrol and 4-hexylresorcinol against oxidative DNA damage in human lymphocytes induced by hydrogen peroxide were investigated. Resveratrol and 4-hexylresorcinol showed no cytotoxicity to human lymphocytes at the tested concentration (10-100 μM). In addition, DNA damage in human lymphocytes induced by H 2 O 2 was inhibited by resveratrol and 4-hexylresorcinol. Resveratrol and 4-hexylresorcinol at concentrations of 10-100 μM induced an increase in glutathione (GSH) levels in a concentration-dependent manner. Moreover, these two compounds also induced activity of glutathione peroxidase (GPX) and glutathione reductase (GR). The activity of glutathione-S-transferase (GST) in human lymphocytes was induced by resveratrol. Resveratrol and 4-hexylresorcinol inhibited the activity of catalase (CAT). These data indicate that the inhibition of resveratrol and 4-hexylresorcinol on oxidative DNA damage in human lymphocytes induced by H 2 O 2 might be attributed to increase levels of GSH and modulation of antioxidant enzymes (GPX, GR and GST).     

Mechanism of oxidative DNA damage induced by carcinogenic allyl isothiocyanate

Several isothiocyanates have been proposed as promising chemopreventive agents for human cancers. However, it has been reported that allyl isothiocyanate exhibit carcinogenic potential, and benzyl isothiocyanate and phenethyl isothiocyanate have tumor-promoting activities. We investigated whether these isothiocyanates could cause DNA damage, using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Allyl isothiocyanate caused Cu(II)-mediated DNA damage and formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) more strongly than benzyl and phenethyl isothiocyanates. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited Cu(II)-mediated DNA damage by these isothiocyanates, suggesting involvement of H(2)O(2) and Cu(I). Isothiocyanates induced DNA damage frequently at thymine and cytosine residues in the presence of Cu(II). A UV-visible spectroscopic study revealed an association between the generation of superoxide and the yield of SH group from isothiocyanates. Furthermore, the yield of 8-oxodG formation was correlated with their superoxide-generating ability. Allyl isothiocyanate significantly induced 8-oxodG formation in HL-60 cells, but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. We conclude that oxidative DNA damage may play important roles in carcinogenic processes induced by allyl isothiocyanate.     

Mechanism of oxidative DNA damage induced by carcinogenic 4-aminobiphenyl

DNA adduct formation is thought to be a major cause of DNA damage by carcinogenic aromatic amines. We investigated the ability of an aromatic amine, 4-aminobiphenyl (4-ABP) and its N-hydroxy metabolite (4-ABP(NHOH)) to cause oxidative DNA damage, using (32)P-labeled human DNA fragments from the p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. 4-ABP(NHOH) was found to cause Cu(II)-mediated DNA damage, especially at thymine residues. Addition of the endogenous reductant NADH led to dramatic enhancement of this process. Catalase and bathocuproine, a Cu(I)-specific chelator, reduced the amount of DNA damage, suggesting the involvement of H(2)O(2) and Cu(I). 4-ABP(NHOH) dose-dependently induced 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in the presence of Cu(ll) and NADH. 4-ABP(NHOH) conversion to nitrosobiphenyl, as measured by UV-visible spectroscopy, occurred rapidly in the presence of Cu(II), suggesting Cu(II)-mediated autoxidation. Increased amounts of 8-OHdG were found in HL-60 cells compared to the H(2)O(2)-resistant clone HP100 following 4-ABP(NHOH) treatment, further supporting the involvement of H(2)O(2). The present study demonstrates that an N-hydroxy derivative of 4-ABP induces oxidative DNA damage through H(2)O(2) in both a cell-free system and in cultured human cells. We conclude that, in addition to DNA adduct formation, oxidative DNA damage may play an important role in the carcinogenic process of 4-ABP.     

Feselol enhances the cytotoxicity and DNA damage induced by cisplatin in 5637 cells

Transitional cell carcinoma (TCC), which is the most common type of bladder cancer, shows resistance to chemotherapeutic agents due to the overexpression of drug efflux pumps. In this study, the effects of feselol, a sesquiterpene coumarin extracted from Ferula badrakema, on cisplatin cytotoxicity were investigated in 5637 cells, a TCC subline. Cell viability and DNA lesion were evaluated by thiazolyl blue tetrazolium bromide and comet assays, respectively. Feselol had no significant cytotoxic effect in 5637 cells but at 32 microg/mL it increased the cytotoxicity of 1 microg/mL cisplatin by 37% after 24 h. Furthermore, the comet assay revealed that DNA damage induced by cisplatin in 5637 cells is enhanced by 31% when used in combination with feselol. Therefore, feselol might be considered as an effective reversal agent for future in vivo and clinical studies.     

Distinct mechanisms of DNA damage in apoptosis induced by quercetin and luteolin

Quercetin has been reported to have carcinogenic effects. However, both quercetin and luteolin have anti-cancer activity. To clarify the mechanism underlying the carcinogenic effects of quercetin, we compared DNA damage occurring during apoptosis induced by quercetin with that occuring during apoptosis induced by luteolin. Both quercetin and luteolin similarly induced DNA cleavage with subsequent DNA ladder formation, characteristics of apoptosis, in HL-60 cells. In HP 100 cells, an H₂O₂-resistant clone of HL-60 cells, the extent of DNA cleavage and DNA ladder formation induced by quercetin was less than that in HL-60 cells, whereas differences between the two cell types were minimal after treatment with luteolin. In addition, quercetin increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in HL-60 cells but not in HP 100 cells. Luteolin did not increase 8-oxodG formation, but inhibited topoisomerase II (topo II) activity of nuclear extract more strongly than quercetin and cleaved DNA by forming a luteolin-topo II-DNA ternary complex. These results suggest that quercetin induces H₂O₂-mediated DNA damage, resulting in apoptosis or mutations, whereas luteolin induces apoptosis via topo II-mediated DNA cleavage. The H₂O₂-mediated DNA damage may be related to the carcinogenic effects of quercetin.     

Distinct mechanisms of DNA damage in apoptosis induced by quercetin and luteolin

Quercetin has been reported to have carcinogenic effects. However, both quercetin and luteolin have anti-cancer activity. To clarify the mechanism underlying the carcinogenic effects of quercetin, we compared DNA damage occurring during apoptosis induced by quercetin with that occuring during apoptosis induced by luteolin. Both quercetin and luteolin similarly induced DNA cleavage with subsequent DNA ladder formation, characteristics of apoptosis, in HL-60 cells. In HP 100 cells, an H₂O₂-resistant clone of HL-60 cells, the extent of DNA cleavage and DNA ladder formation induced by quercetin was less than that in HL-60 cells, whereas differences between the two cell types were minimal after treatment with luteolin. In addition, quercetin increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in HL-60 cells but not in HP 100 cells. Luteolin did not increase 8-oxodG formation, but inhibited topoisomerase II (topo II) activity of nuclear extract more strongly than quercetin and cleaved DNA by forming a luteolin-topo II-DNA ternary complex. These results suggest that quercetin induces H₂O₂-mediated DNA damage, resulting in apoptosis or mutations, whereas luteolin induces apoptosis via topo II-mediated DNA cleavage. The H₂O₂-mediated DNA damage may be related to the carcinogenic effects of quercetin.     

Determination of DNA damage induced by oxidative stress in hyperlipidemic patients.

In the present paper, we report data on the genotoxic properties of hydrogen peroxide in polymorphonuclear neutrophils (PMNLs) separated from normolipidemic and type II/a hyperlipidemic patients. In all, 15 hyperlipidemic patients (11 female, 4 male, mean age 54.6+/-10.25 years) were involved in the study, and 7 normolipidemic patients (5 female, 2 male, mean age 53.4+/-8.07 years) served as controls. Using the comet assay, there was a significant difference in the degree of DNA damage between the two groups. The visual score characteristic of the degree of DNA damage was 350.97+/-31.31 in the hyperlipidemic group, while it was 289.5+/-29.49 in the control group (P<0.001). In the hyperlipidemic patients, a positive correlation was found between the degree of DNA damage and the basic oxidation of PMNLs (r=0.517), and the superoxide anion production of the cells stimulated with phorbolmiristate acetate (PMA) (r=0.326) and formyl-Met-Leu-Phe (FMLP) (r=0.525) as well. There was a negative correlation between DNA damage and HDL-associated antioxidant paraoxonase (PON) activity (r=-0.469), and the PON/HDL ratio (r=-0.631). No correlation was found between the degree of DNA damage and the plasma concentration of nitric oxide (NO) (r=0.098) and thiobarbituric acid-reactive substances (TBARS) (r=0.061) in hyperlipidemic patients. Our results show that in hyperlipidemic patients there is an increase in lymphocyte DNA damage caused by oxidative stress when compared to normolipidemic individuals as demonstrated by comet assay. Decreased antioxidant capacity in hyperlipidemic patients may play a significant role in this process.     

DNA polymerase beta imbalance increases apoptosis and mutagenesis induced by oxidative stress

Oxidative stress has been proposed to be one of the major causes leading to the accumulation of mutation that is associated with the initiation and progression of cancers. Elevated expression of DNA polymerase beta, an event found in many human tumors, has been shown to generate a mutator phenotype. Here, we demonstrated that overexpression of DNA polymerase beta strengthens the mutagenicity of oxidative damages, concomitantly with a higher cellular sensitivity and increased apoptosis. Deregulated expression of DNA polymerase beta could represent a predisposition factor for mutagenic effects of oxidative stress and thus have implication in the generation and/or evolution of cancer.     

The mechanisms of oxidative DNA damage and apoptosis induced by norsalsolinol, an endogenous tetrahydroisoquinoline derivative associated with Parkinson's disease

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 ³²P-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 H₂O₂ 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.     

Copper-mediated oxidative DNA damage induced by eugenol: possible involvement of O-demethylation

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 [32P]-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 H2O2 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.     

Tempol prevents genotoxicity induced by vorinostat: role of oxidative DNA damage

Vorinostat is a member of histone deacetylase inhibitors, which represents a new class of anticancer agents for the treatment of solid and hematological malignancies. Studies have shown that these drugs induce DNA damage in blood lymphocytes, which is proposed to be due to the generation of oxidative lesions. The increase in DNA damage is sometimes associated with risk of developing secondary cancer. Thus, finding a treatment that limits DNA damage caused by anticancer drugs would be beneficial. Tempol is a potent antioxidant that was shown to prevent DNA damage induced by radiation. In this study, we aimed to investigate the harmful effects of vorinostat on DNA damage, and the possible protective effects of tempol against this damage. For that, the spontaneous frequency of sister chromatid exchanges (SCEs), chromosomal aberrations (CAs), and 8-hydroxy-2-deoxy guanosine (8-OHdG) levels were measured in cultured human lymphocytes treated with vorinostat and/or tempol. The results showed that vorinostat significantly increases the frequency of SCEs, CAs and 8-OHdG levels in human lymphocytes as compared to control. These increases were normalized by the treatment of cells with tempol. In conclusion, vorinostat is genotoxic to lymphocytes, and this toxicity is reduced by tempol. Such results could set the stage for future studies investigating the possible usefulness of antioxidants co-treatment in preventing the genotoxicity of vorinostat when used as anticancer in human.     

Thymidine phosphorylase inhibits apoptosis induced by cisplatin

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP, partially prevents hypoxia-induced apoptosis. TP is expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. TP inhibited a number of steps in the cisplatin-induced apoptotic pathway, activation of caspases 3 and 9 and mitochondrial cytochrome c release. These findings suggest a mechanism by which TP confers resistance to apoptosis induced by cisplatin. Moreover, mutant TP that has no enzymatic activity also suppressed cisplatin-induced apoptosis. These findings indicate that TP has cytoprotective functions against cytotoxic agents which are independent of its enzymatic activity.