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

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 (32)P-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.

Mechanism of oxidative DNA damage induced by capsaicin, a principal ingredient of hot chili pepper.

Although capsaicin exhibits antitumor activity, carcinogenic potential has also been reported. To clarify the mechanism for expression of potential carcinogenicity of capsaicin, we examined DNA damage induced by capsaicin in the presence of metal ion and various kinds of cytochrome P450 (CYP) using 32P-5'-end-labeled DNA fragments. Capsaicin induced Cu(II)-mediated DNA damage efficiently in the presence of CYP1A2 and partially in the presence of 2D6. CYP1A2-treated capsaicin caused double-base lesions at 5'-TG-3', 5'-GC-3' and CG of the 5'-ACG-3' sequence complementary to codon 273, a hotspot of p53 gene. DNA damage was inhibited by catalase and bathocuproine, a Cu(I) chelator, suggesting that reactive species derived from the reaction of H2O2 with Cu(I) participate in DNA damage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine was significantly increased by CYP1A2-treated capsaicin in the presence of Cu(II). Therefore, we conclude that Cu(II)-mediated oxidative DNA damage by CYP-treated capsaicin seems to be relevant for the expression of its carcinogenicity.

Evaluation for safety of antioxidant chemopreventive agents.

Antioxidants are considered as the most promising chemopreventive agents against various human cancers. However, some antioxidants play paradoxical roles, acting as "double-edged sword." A primary property of effective and acceptable chemopreventive agents should be freedom from toxic effects in healthy population. Miscarriage of the intervention by beta-carotene made us realize the necessity for evaluation of safety before recommending use of antioxidant supplements for chemoprevention. We have evaluated the safety of antioxidants on the basis of reactivity with DNA. Our results revealed that phytic acid, luteolin, and retinoic acid did not cause DNA damage under the experimental condition. Furthermore, phytic acid inhibited the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, in cultured cells treated with a H(2)O(2)-generating system. Thus, it is expected that these chemopreventive agents can safely protect humans against cancer. On the other hand, some chemopreventive agents with prooxidant properties (alpha-tocopherol, quercetin, catechins, isothiocyanates, N-acetylcysteine) caused DNA damage via generation of reactive oxygen species in the presence of metal ions and endogenous reductants under some circumstances. Furthermore, other chemopreventive agents (beta-carotene, genistein, daidzein, propyl gallate, curcumin) exerted prooxidant properties after metabolic activation. Therefore, further studies on safety should be required when antioxidants are used for cancer prevention.

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.

Oxidative DNA damage induced by a melatonin metabolite, 6-hydroxymelatonin, via a unique non-o-quinone type of redox cycle.

Melatonin, an indolic pineal hormone, is produced primarily at night in mammals and is important in controlling biological rhythms. Although melatonin is known to be effective as a free radical scavenger and has an anti-cancer effect, carcinogenic properties have also been reported. In relation to its carcinogenic potential, we have examined whether 6-hydroxymelatonin, a major melatonin metabolite, can induce DNA damage in the presence of metal ion using [32P]-5'-end-labeled DNA fragments obtained from genes relevant to human cancer. 6-Hydroxymelatonin induced site-specific DNA damage in the presence of Cu(II). Formamidopyrimidine-DNA glycosylase treatment induced cleavage sites mainly at G residues of the 5'-TG-3' sequence, whereas piperidine treatment induced cleavage sites at T mainly of 5'-TG-3'. Interestingly, 6-hydroxymelatonin strongly damaged G and C of the 5'-ACG-3' sequence complementary to codon 273 of the p53 gene. These results suggest that 6-hydroxymelatonin can cause double-base lesions. DNA damage was inhibited by both catalase and bathocuproine, Cu(I)-specific stabilizer, suggesting that reactive species derived from the reaction of H2O2 with Cu(I) participate in DNA damage. Cytochrome P450 reductase efficiently enhanced 6-hydroxymelatonin-induced oxidative DNA damage and oxygen consumption, suggesting the formation of redox cycle. It is noteworthy that 6-hydroxymelatonin can efficiently induce DNA damage via non-o-quinone type of redox cycle. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in calf thymus DNA was significantly increased by 6-hydroxymelatonin in the presence of Cu(II). Furthermore, 6-hydroxymelatonin significantly increased the formation of 8-oxodG in human leukemia cell line HL-60 but not in HP100, a hydrogen peroxide (H2O2)-resistant cell line derived from HL-60. The 6-hydroxymelatonin-induced 8-oxodG formation in HL-60 cells significantly decreased by the addition of bathocuproine or o-phenanthroline. Therefore, it is concluded that melatonin may exhibit carcinogenic potential through oxidative DNA damage by its metabolite.

Oxidative DNA damage induced by benz[a]anthracene metabolites via redox cycles of quinone and unique non-quinone.

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.

Molecular mechanisms of DNA damage induced by procarbazine in the presence of Cu(II).

Procarbazine [N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide], a hydrazine derivative, which has been shown to have effective antineoplastic activity, induces cancer in some experimental animals and humans. To clarify a new mechanism for its carcinogenic effect, we examined DNA damage induced by procarbazine in the presence of metal ion, using 32P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Procarbazine plus Cu(II) induced piperidine-labile and formamidopyrimidine-DNA glycosylase-sensitive lesions at the 5'-ACG-3' sequence, complementary to a hotspot of the p53 gene, and the 5'-TG-3' sequence. Catalase partially inhibited DNA damage, suggesting that not only H(2)O(2) but also other reactive species are involved. Procarbazine plus Cu(II) significantly increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, which was completely inhibited by calatase. Electron spin resonance spin-trapping experiments revealed that methyl radicals were generated from procarbazine and Cu(II). On the basis of these findings, it is considered that procarbazine causes DNA damage through non-enzymatic formation of the Cu(I)-hydroperoxo complex and methyl radicals. In conclusion, in addition to alkylation, oxidative DNA damage may play important roles in not only antitumor effects but also mutagenesis and carcinogenesis induced by procarbazine.

Sequence-specific DNA damage induced by ultraviolet A-irradiated folic acid via its photolysis product.

DNA damage mediated by photosensitizers participates in solar carcinogenesis. Fluorescence measurement and high-performance liquid chromatography analysis demonstrated that photoirradiated folic acid, one of the photosensitizers in cells, generates pterine-6-carboxylic acid (PCA). Experiments using 32P-labeled DNA fragments obtained from a human gene showed that ultraviolet A-irradiated folic acid or PCA caused DNA cleavage specifically at consecutive G residues in double-stranded DNA after Escherichia coli formamidopyrimidine-DNA glycosylase or piperidine treatment. The amount of 8-oxo-7,8-dihydro-2(')-deoxyguanosine formed through this DNA photoreaction in double-stranded DNA exceeded that in single-stranded DNA. Kinetic studies suggested that DNA damage is caused mainly by photoexcited PCA generated from folic acid rather than by folic acid itself. In conclusion, photoirradiated folic acid generates PCA, which induces DNA photooxidation specifically at consecutive G residues through electron transfer. Excess intake of folic acid supplements may increase a risk of skin cancer by solar ultraviolet light.