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.     

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.     

Metal-mediated DNA damage induced by curcumin in the presence of human cytochrome P450 isozymes

Although curcumin is known to exhibit antitumor activity, carcinogenic properties have also been reported. To clarify the potentiality of carcinogenesis by curcumin, we have examined whether curcumin can induce DNA damage in the presence of cytochrome P450 (CYP) using [32P]-5(')-end-labeled DNA fragments obtained from genes relevant to human cancer. Curcumin treated with CYP 2D6, CYP1A1, or CYP1A2 induced DNA damage in the presence of Cu(II). CYP2D6-treated curcumin caused base damage, especially at 5(')-TG-3('), 5(')-GC-3('), and GG sequences. The DNA damage was inhibited by both catalase and bathocuproine, suggesting that reactive species derived from the reaction of H(2)O(2) with Cu(I) participate in DNA damage. Formation of 8-oxo-7,8-dihydro-2(')-deoxyguanosine was significantly increased by CYP2D6-treated curcumin in the presence of Cu(II). Time-of- flight mass spectrometry demonstrated that CYP2D6 catalyzed the conversion of curcumin to O-demethyl curcumin. Therefore, it is concluded that curcumin may exhibit carcinogenic potential through oxidative DNA damage by its metabolite.     

Sequence-specific DNA damage induced by carcinogenic danthron and anthraquinone in the presence of Cu(II), cytochrome P450 reductase and NADPH

The mechanism of metal-mediated DNA damage by carcinogenic danthron (1,8-dihydroxyanthraquinone) and anthraquinone was investigated by the DNA sequencing technique using ³²P-labeled human DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Danthron caused DNA damage particularly at guanines in the 5'-GG-3', 5-GGGG-3', 5'-GGGGG-3' sequences (damaged bases are underlined) in the presence of Cu(II), cytochrome P450 reductase and the NADPH-generating system. 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 increased with increasing concentration of danthron. On the other hand, carcinogenic anthraquinone induced less oxidative DNA damage than danthron. Electron spin resonance study showed that the semiquinone radical could beproduced by P450 reductase plus NADPH-mediated reduction of danthron, while little signal was observed with anthraquinone. These results suggest that danthron is much more likely to be reduced by P450 reductase and generate reactive oxygen species through the redox cycle, leading to more extensive Cu(II)-mediated DNA damage than anthraquinone. In the case of anthraquinone, its hydroxylated metabolites with similar reactivity to danthron may participate in DNA damage in vivo. We conclude that oxidative DNA damage by danthron and anthraquinone seems to be relevant for the expression of their carcinogenicity.     

Mechanism of metal-mediated DNA damage induced by metabolites of carcinogenic 2-nitropropane

2-Nitropropane (2-NP), a widely used industrial solvent, is carcinogenic to rats. To clarify the mechanism of carcinogenesis by 2-NP, we investigated DNA damage by 2-NP metabolites, N-isopropylhydroxylamine (IPHA) and hydroxylamine-O-sulfonic acid (HAS), using 32P-5'-end-labelled DNA fragments obtained from genes that are relevant to human cancer. In the presence of Fe(III) EDTA, both IPHA and HAS caused DNA damage at every nucleotide position without marked site preference. The damage was inhibited by free hydroxyl radical (-*OH) scavengers, catalase and deferoxamine mesilate, an iron chelating agent. These results suggest that the DNA damage was caused by -*OH generated via H(2)O(2) by both IPHA and HAS. In contrast, in the presence of Cu(II), IPHA frequently caused DNA damage at thymine. The Cu(II)-mediated DNA damage caused by IPHA was inhibited by catalase, methional and bathocuproine, a Cu(I)-specific chelator, suggesting the involvement of H(2)O(2) and Cu(I). These results suggest that the DNA damage induced by IPHA in the presence of Cu(II) was caused by a reactive oxygen species like the Cu(I)-hydroperoxo complex. On the other hand, HAS most frequently induced DNA damage at 5'-TG-3', 5'-GG-3' and 5'-GGG-3' sequences. Catalase and methional only partly inhibited the Cu(II)-mediated DNA damage caused by HAS, suggesting that the reactive oxygen species and another reactive species participate in this process. Formation of 8-oxodG by IPHA or HAS increased in the presence of metal ions. This study suggests that metal-mediated DNA damage caused by 2-NP metabolites plays an important role in the mutagenicity and the carcinogenicity of 2-NP.     

Copper-dependent DNA damage induced by hydrazobenzene, an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5'-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Photosensitized DNA damage induced by NADH: site specificity and mechanism

Increasing evidence reveals the carcinogenicity of UVA radiation. We demonstrated that UVA-irradiated NADH induced damage to (32)P-labeled DNA fragments obtained from the p53 gene in the presence of Cu(II). Formamidopyrimidine glycosylase (Fpg)-sensitive lesions were formed at guanine residues, whereas piperidine-labile lesions occurred frequently at thymine residues. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), upon UVA exposure in the presence of Cu(II), increased depending on NADH concentration. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited the DNA damage, suggesting the involvement of reactive species derived from H(2)O(2) and Cu(I). UVA-irradiated riboflavin induced DNA cleavage through electron transfer at 5' guanine of the 5'-GG-3' sequence with both Fpg and piperidine treatments; Fpg induced less cleavage at the guanine residues than piperidine. These results imply that NADH may participate as an endogenous photosensitizer in UVA carcinogenesis via H(2)O(2) generation, producing metal-mediated mutagenic lesions such as 8-oxodG.     

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.     

Double base lesions of DNA by a metabolite of carcinogenic benzo[a]pyrene.

Carcinogenic benzo[a]pyrene (BP) is generally considered to show genotoxicity by forming DNA adducts of its metabolite, BP-7,8-diol-9,10-epoxide. We investigated oxidative DNA damage and its sequence specificity induced by BP-7,8-dione, another metabolite of BP, using (32)P-5'-end-labeled DNA. Formamidopyrimidine-DNA glycosylase treatment induced cleavage sites mainly at G residues of 5'-TG-3' sequence and at poly(C) sequences, in DNA incubated with BP-7,8-dione in the presence of NADH and Cu(II), whereas piperidine treatment induced cleavage sites at T mainly of 5'-TG-3'. BP-7,8-dione strongly damaged the G and C of the ACG sequence complementary to codon 273 of the p53 gene. Catalase and a Cu(I)-specific chelator attenuated the DNA damage, indicating the involvement of H(2)O(2) and Cu(I). BP-7,8-dione with NADH and Cu(II) also increased 8-oxo-7,8-dihydro-2'-deoxyguanosine formation. We conclude that oxidative DNA damage, especially double base lesions, may participate in the expression of carcinogenicity of BP in addition to DNA adduct formation.     

Radical production and DNA damage induced by carcinogenic 4-hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus

4-Hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus, is carcinogenic to rodents. To clarify the mechanism of carcinogenesis, we investigated DNA damage by 4-hydrazinobenzoic acid using ³²P-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes. 4-Hydrazinobenzoic acid induced Cu(II)-dependent DNA damage especially piperidine-labile formation at thymine and cytosine residues. Typical hydroxyl radical scavengers showed no inhibitory effects on Cu(II)-mediated DNA damage by 4-hydrazinobenzoic acid. Bathocuproine and catalase inhibited the DNA damage, indicating the participation of Cu(I) and H₂O₂ in the DNA damage. These findings suggest that H₂O₂ generated by the autoxidation of 4-hydrazinobenzoic acid reacts with Cu(I) to form reactive oxygen species, capable of causing DNA damage. Interestingly, catalase did not completely inhibit DNA damage caused by a high concentration of 4-hydrazinobenzoic acid (over 50 μM) in the presence of Cu(II). 4-Hydrazinobenzoic acid induced piperidine-labile sites frequently at adenine and guanine residues in the presence of catalase. 4-Hydrazinobenzoic acid increased formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in calf thymus DNA, whereas 4-hydrazinobenzoic acid did not increase the formation of 8-oxodG in the presence of catalase. ESR spin-trapping experiments showed that the phenyl radical was formed during the reaction of 4-hydrazinobenzoic acid in the presence of Cu(II) and catalase. Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF/mass) spectrometry analysis showed that phenyl radical formed adduct with adenosine and guanosine. These results suggested that 4-hydrazinobenzoic acid induced DNA damage via not only H₂O₂ production but also phenyl radical production. This study suggests that both oxidative DNA damage and DNA adduct formation play important roles in the expression of carcinogenesis of 4-hydrazinobenzoic acid.     

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 a metabolite of carcinogenic and reproductive toxic nitrobenzene in the presence of NADH and Cu(II)

The mechanism of DNA damage induced by metabolites of nitrobenzene was investigated in relation to the carcinogenicity and reproductive toxicity of nitrobenzene. Nitrosobenzene, a nitrobenzene metabolite, induced NADH plus Cu(II)-mediated DNA cleavage frequently at thymine and cytosine residues. Catalase and bathocuproine inhibited the DNA damage, suggesting the involvement of H2O2 and Cu(I). Typical free hydroxyl radical scavengers showed no inhibitory effects on DNA damage. Nitrosobenzene caused the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of NADH and Cu(II). ESR spectroscopic study has confirmed that nitrosobenzene is reduced by NADH to the phenylhydronitroxide radical even in the absence of Cu(II). These results suggest that nitrosobenzene can be reduced non-enzymatically by NADH, and the redox cycle reaction resulted in oxidative DNA damage due to the copper-oxygen complex, derived from the reaction of Cu(I) with H2O2.     

High yield of endoreduplication induced by ICRF-193: a topoisomerase II catalytic inhibitor

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 phi X-174 plasmid DNA. H(2)O(2)/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 H(2)O(2)/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H(2)O(2) 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 H(2)O(2)/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 H(2)O(2) 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.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Metal-mediated oxidative damage to cellular and isolated DNA by gallic acid, a metabolite of antioxidant propyl gallate

Propyl gallate (PG), widely used as an antioxidant in foods, is carcinogenic to mice and rats. PG increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60, but not in HP100, which is hydrogen peroxide (H2O2)-resistant cell line derived from HL-60. Although PG induced no or little damage to 32P-5'-end-labeled DNA fragments obtained from genes that are relevant to human cancer, DNA damage was observed with treatment of esterase. HPLC analysis of the products generated from PG incubated with esterase revealed that PG converted into gallic acid (GA). GA induced DNA damage in a dose-dependent manner in the presence of Fe(III)EDTA or Cu(II). In the presence of Fe(III) complex such as Fe(III)EDTA or Fe(III)ADP, GA caused DNA damage at every nucleotide. Fe(III) complex-mediated DNA damage by GA was inhibited by free hydroxy radical (*OH) scavengers, catalase and an iron chelating agent. These results suggested that the Fe(III) complex-mediated DNA damage caused by GA is mainly due to *OH generated via the Fenton reaction. In the presence of Cu(II), DNA damage induced by GA occurred at thymine and cytosine. Although *OH scavengers did not prevent the DNA damage, methional inhibited the DNA damage. Cu(II)-mediated DNA damage was inhibited by catalase and a Cu(I) chelator. These results indicated that reactive oxygen species formed by the interaction of Cu(I) and H2O2 participates in the DNA damage. GA increased 8-oxodG content in calf thymus DNA in the presence of Cu(II), Fe(III)EDTA or Fe(III)ADP. This study suggested that metal-mediated DNA damage caused by GA plays an important role in the carcinogenicity of PG.     

Catechins induce oxidative damage to cellular and isolated DNA through the generation of reactive oxygen species

Green tea catechins have antimutagenic and anticarcinogenic activities. On the other hand, several epidemiological studies have indicated significant positive relationship between green tea consumption and cancer. Catechins enhance colon carcinogenesis in rats initiated with chemical carcinogen. To clarify the mechanism underlying the potential carcinogenicity, we investigated the DNA-damaging ability of catechins in human cultured cells. Catechin increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60 but not in HP100, a hydrogen peroxide (H

2

O

2

)-resistant cell line derived from HL-60. The catechin-induced formation of 8-oxodG in HL-60 cells significantly decreased by bathocuproine. Furthermore, we investigated DNA damage and its site-specificity induced by catechins, using

32

P-labeled DNA fragments. Catechin and epicatechin induced extensive DNA damage in the presence of Cu(II). Catechin caused piperidine-labile sites at thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine inhibited the DNA damage, indicating the involvement of H

2

O

2

and Cu(I). NADH enhanced catechins plus Cu(II)-induced 8-oxodG formation in calf thymus DNA, suggesting the redox cycle between catechins and their corresponding quinones, the oxidized forms of catechins. The DNA-damaging ability of epicatechin is stronger than that of catechin, possibly due to the greater turnover frequency of the redox cycle. The difference in their redox properties could be explained by their redox potentials estimated form an ab initio molecular orbital calculation. The present study demonstrated that catechins could induce metal-dependent H

2

O

2

generation during the redox reactions and subsequently damage to cellular and isolated DNA. Therefore, it is reasonably considered that green tea catechins may have the dual function of anticarcinogenic and carcinogenic potentials.     

Distinct mechanisms of oxidative DNA damage by two metabolites of carcinogenic o-toluidine

Mechanisms of DNA damage by metabolites of carcinogenic o-toluidine in the presence of metals were investigated by the DNA sequencing technique using (32)P-labeled human DNA fragments. 4-Amino-3-methylphenol, a major metabolite, caused DNA damage in the presence of Cu(II). Predominant cleavage sites were thymine and cytosine residues. o-Nitrosotoluene, a minor metabolite, did not induce DNA damage even in the presence of Cu(II), but addition of NADH induced DNA damage very efficiently. The DNA cleavage pattern was similar to that in the case of 4-amino-3-methylphenol. Bathocuproine and catalase inhibited DNA damage by these o-toluidine metabolites, indicating the participation of Cu(I) and H(2)O(2) in the DNA damage. Typical free hydroxyl radical scavengers showed no inhibitory effects on the DNA damage. o-Toluidine metabolites increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in calf thymus DNA in the presence of Cu(II). UV-visible and ESR spectroscopic studies have demonstrated that 4-amino-3-methylphenol is autoxidized to form the aminomethylphenoxyl radical and o-nitrosotoluene is reduced by NADH to the o-toluolhydronitroxide radical in the presence and absence of Cu(II). Consequently, it is considered that these radicals react with O(2) to form O(-)(2) and subsequently H(2)O(2), and that the reactive species generated by the reaction of H(2)O(2) with Cu(I) participate in the DNA damage. Metal-mediated DNA damage by o-toluidine metabolites through H(2)O(2) seems to be relevant for the expression of the carcinogenicity of o-toluidine.     

Carcinogenic 3-nitrobenzanthrone induces oxidative damage to isolated and cellular DNA

3-Nitrobenzanthrone (3-NBA) is an extremely potent mutagen in diesel exhaust. It is a lung carcinogen to rats, and therefore a suspected carcinogen to human. In order to clarify the mechanism of carcinogenicity of 3-NBA, we investigated oxidative DNA damage by N-hydroxy-3-aminobenzanthrone (N-OH-ABA), a metabolite of 3-NBA, using 32P-labeled DNA fragments from the human p53 tumor-suppressor gene. N-OH-ABA caused Cu(II)-mediated DNA damage, and endogenous reductant NADH dramatically enhanced this process. Catalase and a Cu(I)-specific chelator decreased DNA damage, suggesting the involvement of hydrogen peroxide (H2O2) and Cu(I). N-OH-ABA induced DNA damage at cytosine and guanine residues of ACG sequence complementary to codon 273, a well-known hot spot of the p53 gene. N-OH-ABA dose dependently induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in the presence of Cu(II) and NADH. Treatment with N-OH-ABA increased amounts of 8-oxodG in HL-60 cells compared to the H2O2-resistant clone HP100, supporting the involvement of H2O2. The present study has demonstrated that the N-hydroxy metabolite of 3-NBA induces oxidative DNA damage through H2O2 in both a cell-free system and cultured human cells. We conclude that oxidative DNA damage may play an important role in the carcinogenic process of 3-NBA in addition to previously reported DNA adduct formation.     

Carcinogenic semicarbazide induces sequence-specific DNA damage through the generation of reactive oxygen species and the derived organic radicals

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.     

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.