phosphorus-radioisotopes and bathocuproine

Polychlorinated biphenyls (PCBs) are toxic industrial chemicals, complete carcinogens, and efficacious tumor promoters. However, the mechanism(s) of PCB-mediated carcinogenicity remains largely undefined. One likely pathway by which these agents may play a role in carcinogenesis is the generation of oxidative DNA damage by redox cycling of dihydroxylated PCB metabolites. We have now employed a new (32)P-postlabeling system to examine novel oxidative DNA lesions induced by Cu(2+)-mediated activation of PCB metabolites. (32)P postlabeling of DNA incubated with various PCB metabolites resulted in over a dozen novel polar oxidative DNA adducts that were chromatographically similar for all active agents. The most potent metabolites tested were the hydroquinones (hydroxyl groups arranged para to each other), yielding polar oxidative adduct levels ranging from 55 to 142 adducts/10(6) nucleotides. PCB catechols, or ortho-dihydroxy metabolites, were up to 40% less active than their corresponding hydroquinone congeners, whereas monohydroxylated and quinone metabolites did not produce detectable oxidative damage over that of vehicle. With the exception of 2,4,5-Cl-2',5'-dihydroxybiphenyl, this oxidative DNA damage seemed to be inversely related to chlorine content: no chlorine approximately mono->di->trichlorinated metabolites. Importantly, copper, but not iron, was essential for activation of the PCB metabolites to these polar oxidative DNA adducts, because in its absence or in the presence of the Cu(+)-specific scavenger bathocuproine, no adducts were detected. Intervention studies with known reactive oxygen species (ROS) modifiers suggested that H(2)O(2), singlet oxygen, hydroxyl radical, and superoxide may also be involved in this PCB-mediated oxidative DNA damage. These data indicate a mechanistic role for several ROS, in addition to copper, in PCB-induced DNA damage and provide further support for oxidative DNA damage in PCB-mediated carcinogenesis.

Topics: Animals; Carcinogenicity Tests; Carcinogens, Environmental; Copper; DNA; DNA Adducts; Hydroquinones; Hydroxylation; In Vitro Techniques; Mixed Function Oxygenases; Neoplasms; Oxidative Stress; Phenanthrolines; Phosphorus Radioisotopes; Polychlorinated Biphenyls; Reactive Oxygen Species; Salmon

Semicarbazide, a hydrazine derivative, is carcinogenic to mice but shows no or little mutagenicity in the Salmonella-microsome test. To clarify whether or not the genotoxic mechanism contributes to the non-mutagenic carcinogenicity of semicarbazide, we investigated DNA damage induced by semicarbazide using 32P-5'-end-labeled DNA fragments obtained from the c-Ha-ras-1 protooncogene and the p53 tumor suppressor gene. Semicarbazide caused DNA damage frequently at the thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine partially inhibited DNA damage, suggesting that hydrogen peroxide plus Cu(I) participates in DNA damage. When a high concentration of semicarbazide was used in the presence of catalase, DNA damage was induced, especially at G in 5'-AG and slightly at 5'-G in GG and GGG sequences. An electron paramagnetic resonance (EPR) spectroscopic study has confirmed that the reaction of semicarbazide with Cu(II) produces carbamoyl radicals (z.rad;CONH(2)), possibly generated via the nitrogen-centered radicals of semicarbazide. Azodicarbonamide also produced carbamoyl radicals and induced DNA damage frequently at 5'-G in GG and GGG sequences, suggesting that carbamoyl radicals participate in this sequence-specific DNA damage by semicarbazide. On the basis of our previous reports, we consider that the sequence-specific DNA damage at G in 5'-AG in the present study is due to the nitrogen-centered radicals. This study has shown that semicarbazide induces DNA damage in the presence of Cu(II) through the formation of hydrogen peroxide and Cu(I). In addition, semicarbazide-derived free radicals participate in DNA damage. DNA damage induced by these reactive species may be relevant to the carcinogenicity of semicarbazide.

Topics: Animals; Carcinogens; Cattle; Chromatography, High Pressure Liquid; Copper; Deoxyguanosine; DNA; DNA Damage; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Genes, p53; Genes, ras; Guanosine; Phenanthrolines; Phosphorus Radioisotopes; Promoter Regions, Genetic; Reactive Oxygen Species; Semicarbazides; Substrate Specificity; Thymus Gland

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

Topics: Animals; Benz(a)Anthracenes; Catalase; Copper; Deoxyadenosines; DNA Damage; DNA Fragmentation; Dose-Response Relationship, Drug; Genes, p16; Genes, ras; Guanine; Humans; NAD; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Oxidative Stress; Phenanthrolines; Phosphorus Radioisotopes; Polycyclic Aromatic Hydrocarbons; Quinones; Tumor Suppressor Protein p53

Estrogen-induced carcinogenesis involves enhanced cell proliferation (promotion) and genotoxic effects (initiation). To investigate the contribution of estrogens and their metabolites to tumor initiation, we examined DNA damage induced by estradiol and its metabolites, the catechol estrogens 2-hydroxyestradiol (2-OHE(2)) and 4-hydroxyestradiol (4-OHE(2)). In the presence of Cu(II), catechol estrogens formed piperidine-labile sites at thymine and cytosine residues in (32)P 5'-end-labeled DNA fragments and induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine. NADH markedly enhanced Cu(II)-dependent DNA damage mediated by nanomolar concentrations of catechol estrogens. Catalase and bathocuproine inhibited the DNA damage, suggesting the involvement of H(2)O(2) and Cu(I). These results suggest that H(2)O(2), generated during Cu(II)-catalyzed autoxidation of catechol estrogens, reacts with Cu(I) to form the Cu(I)-peroxide complex, leading to oxidative DNA damage, and that NADH enhanced DNA damage through the formation of redox cycle. To investigate the role of estrogens and their metabolites in tumor promotion, we examined their effects on proliferation of estrogen-dependent MCF-7 cells. Estradiol enhanced the proliferation of MCF-7 cells at much lower concentrations than catechol estrogens. These findings indicate that catechol estrogens play a role in tumor initiation through oxidative DNA damage, whereas estrogens themselves induce tumor promotion and/or progression by enhancing cell proliferation in estrogen-induced carcinogenesis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Cell Division; Copper; Deoxyguanosine; DNA; DNA Damage; DNA Fragmentation; Drug Interactions; Estradiol; Estrogens, Catechol; Free Radical Scavengers; Humans; NAD; Oxidative Stress; Phenanthrolines; Phosphorus Radioisotopes; Tumor Cells, Cultured

Nitro derivative (nitro-IQ) of a carcinogenic heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is known to be a potent mutagen as well as IQ, and nitro-IQ is believed to be activated enzymatically by nitroreductase. We investigated nonenzymatic reduction of nitro-IQ by an endogenous reductant NADH and the ability of inducing DNA damage by nitro-IQ. Nitro-IQ caused DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of NADH and Cu(II). Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited the DNA damage, suggesting the involvement of H2O2 and Cu(I). Nitro-IQ induced DNA cleavage frequently at thymine and cytosine residues in the presence of NADH and Cu(II). UV-vis spectroscopic study showed that no spectral change of Nitro-IQ and NADH was observed in the absence of Cu(II), while rapid spectral change was observed in the presence of Cu(II), suggesting that Cu(II) mediated redox reaction of nitro-IQ and NADH. These results suggest that nitro-IQ can be reduced nonenzymatically by NADH in the presence of Cu(II), and the redox reaction resulted in oxidative DNA damage due to the copper-oxygen complex, derived from the reaction of Cu(I) with H2O2. We conclude that nonenzymatic reduction of nitro-IQ and resulting in oxidative DNA damage can play a role in carcinogenesis of IQ.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Cattle; Copper; Deoxyguanosine; DNA; DNA Damage; Free Radical Scavengers; Humans; Mutagens; NAD; Oxidation-Reduction; Oxidative Stress; Phenanthrolines; Phosphorus Radioisotopes; Proto-Oncogene Proteins p21(ras); Quinolines; Spectrophotometry, Ultraviolet; Tumor Suppressor Protein p53

Benzoyl peroxide (BzPO), a free-radical generator, has tumor-promoting activity. As a method for approaching the mechanism of tumor promoter function, the ability of oxidative DNA damage by BzPO was investigated by using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. BzPO induced piperidine-labile sites at the 5'-site guanine of GG and GGG sequences of double-stranded DNA in the presence of Cu(I), whereas the damage occurred at single guanine residues of single-stranded DNA. Both methional and dimethyl sulfoxide (DMSO) inhibited DNA damage induced by BzPO and Cu(I), but typical hydroxyl radical ((*)OH) scavengers, superoxide dismutase (SOD) and catalase, did not inhibit it. On the other hand, H(2)O(2) induced piperidine-labile sites at cytosine and thymine residues of double-stranded DNA in the presence of Cu(I). Phenylhydrazine, which is known to produce phenyl radicals, induced Cu(I)-dependent damage at thymine residues but not at guanine residues. These results suggest that the BzPO-derived reactive species causing DNA damage is different from (*)OH and phenyl radicals generated from benzoyloxyl radicals. BzPO/Cu(I) induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in double-stranded DNA more effectively than that in single-stranded DNA. Furthermore, we observed that BzPO increased the amount of 8-oxodG in human cultured cells. Consequently, it is concluded that benzoyloxyl radicals generated by the reaction of BzPO with Cu(I) may oxidize the 5'-guanine of GG and GGG sequences in double-stranded DNA to lead to 8-oxodG formation and piperidine-labile guanine lesions, and the damage seems to be relevant to the tumor-promoting activity of BzPO.

Topics: 5' Untranslated Regions; 8-Hydroxy-2'-Deoxyguanosine; Animals; Base Sequence; Benzoyl Peroxide; Carcinogens; Cattle; Clone Cells; Copper; Deoxyguanosine; DNA; DNA Damage; Free Radical Scavengers; Guanine; HL-60 Cells; Humans; Hydrogen Peroxide; Phenanthrolines; Phosphorus Radioisotopes; Thymus Gland

To clarify the mechanism of carcinogenesis by hair dyes, we compared the extent of DNA damage induced by mutagenic m-phenylenediamine and 4-methoxy-m-phenylenediamine, using 32P-5'-end-labeled DNA fragments obtained from the human c-Ha-ras-1 protooncogene and the p53 tumor suppressor gene. Carcinogenic 4-methoxy-m-phenylenediamine caused DNA damage at thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited 4-methoxy-m-phenylenediamine-induced DNA damage, suggesting the involvement of H2O2 and Cu(I). Superoxide dismutase (SOD) enhanced the DNA damage. Formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) was induced by 4-methoxy-m-phenylenediamine in the presence of Cu(II). UV-visible spectroscopic studies have shown that Cu(II) mediated autoxidation of 4-methoxy-m-phenylenediamine and SOD accelerated the autoxidation. On the other hand, non-carcinogenic m-phenylenediamine did not cause clear DNA damage and significant autoxidation even in the presence of Cu(II). These results suggest that carcinogenicity of m-phenylenediamines is associated with ability to cause oxidative DNA damage rather than bacterial mutagenicity.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Carcinogens; Chromatography, High Pressure Liquid; Copper; Cytochrome c Group; Deoxyguanosine; DNA; DNA Damage; Electrochemistry; Free Radical Scavengers; Genes, p53; Genes, ras; Humans; Phenanthrolines; Phenylenediamines; Phosphorus Radioisotopes; Reactive Oxygen Species; Spectrophotometry, Ultraviolet

The tryptophan metabolites 3-hydroxyanthranilic acid (3-HAA) and 3-hydroxykynurenine (3-HKyn) are carcinogens. DNA damage by 3-HAA and 3-HKyn in the presence of metal ions was investigated as a potential mechanism of their carcinogenicity. Pulsed field gel electrophoresis showed that in the presence of Mn(II), 3-HAA and 3-HKyn induced DNA double-strand breaks in cultured human cells. DNA single-strand breaks were observed with alkali treatment. The enhancing effect of catalase inhibitor and the inhibitory effect of o-phenanthroline on the strand breakage indicated the involvement of H2O2 and endogenous transition metal ion. Damage to DNA fragments obtained from c-Ha-rds-1 protooncogene was investigated by a DNA sequencing technique. 3-HAA and 3-HKyn induced piperidine-labile sites frequently at thymine and guanine residues in the presence of Cu(II). The inhibitory effects of bathocuproine and catalase on Cu(II)-mediated DNA damage suggest that Cu(I) and H2O2 have important roles in the production of active species causing DNA damage. The Cu(II)-mediated DNA damage was enhanced by preincubation of 3-HAA with Mn(II). UV-visible spectroscopy showed that Mn(II) and Cu(II) enhanced the rate of autoxidation of 3-HAA in different ways. These results suggest that in the presence of Mn(II) or Cu(II), these tryptophan metabolites produce H2O2, which is activated by transition metal ion to cause damage to DNA both in the case of isolated DNA and cultured cells.

Topics: 3-Hydroxyanthranilic Acid; Animals; Autoradiography; Base Sequence; Carcinogens; Cattle; Cells, Cultured; Copper; DNA; DNA Damage; Free Radical Scavengers; Genes, ras; Humans; Indicators and Reagents; Kynurenine; Manganese Poisoning; Molecular Sequence Data; Oxidation-Reduction; Phenanthrolines; Phosphorus Radioisotopes; Spectrophotometry, Ultraviolet; Tryptophan

Pentachlorophenol (PCP) has been shown to be carcinogenic for mice, although it does not seem to be mutagenic in bacterial test systems. In this study, the mechanism of DNA damage by PCP metabolites in the presence of metals was investigated with a DNA sequencing technique using 32P-labeled DNA fragments and with an electrochemical detector coupled to an HPLC. The metabolite tetrachlorohydroquinone (TCHQ) caused DNA damage in the presence of Cu(II) but not in the presence of either Mn(II) or Fe(III). TCHQ plus Cu(II) frequently induced piperidine-labile sites at thymine residues and guanine residues. The most preferred sites were the thymine residues of the 5'-GTC-3' sequence. TCHQ increased 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). Typical OH scavengers showed no inhibitory effects on TCHQ- plus Cu(II)-induced DNA damage. Bathocuproine and catalase inhibited DNA damage, suggesting that Cu(I) and H2O2 have important roles in the production of active species causing DNA damage. Tetrachloro-p-benzoquinone (TCBQ) alone did not induce DNA damage in the presence of Cu(II), but addition of NADH induced DNA cleavage even in the absence of NADH-FMN oxidoreductase. UV-visible and ESR spectroscopies have demonstrated that TCHQ is rapidly autoxidized into semiquinone even in the absence of metal ions, indicating that the semiquinone radical itself is not the main active species inducing DNA damage. These results suggest that the semiquinone radical produced by the autoxidation of TCHQ and/or the reduction of TCBQ by NADH reacts with dioxygen to form superoxide and subsequently H2O2, which is activated by transition metals to cause DNA damage.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Base Sequence; Cattle; Copper; Deoxyguanosine; DNA; DNA Damage; Free Radical Scavengers; Hydroquinones; Molecular Sequence Data; Mutagens; NAD; Oxidation-Reduction; Pentachlorophenol; Phenanthrolines; Phosphorus Radioisotopes; Proto-Oncogene Proteins p21(ras); Reactive Oxygen Species; Thymus Gland