Site specificity and mechanism of oxidative DNA damage induced by carcinogenic catechol
Shinji Oikawa,
Iwao Hirosawa1,,
Kazutaka Hirakawa2, and
Shosuke Kawanishi3,
Department of Hygiene and
2 Radioisotope Center, Mie University School of Medicine, Mie 514-8507 and
1 Department of Hygiene, Yamaguchi University School of Medicine, Yamaguchi 755-8505, Japan
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Abstract
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Catechol, a naturally occurring and an important industrial chemical, has been shown to have strong promotion activity and induce glandular stomach tumors in rodents. In addition, catechol is a major metabolite of carcinogenic benzene. To clarify the carcinogenic mechanism of catechol, we investigated DNA damage using human cultured cell lines and 32P-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c-Ha-ras-1 proto-oncogene. Catechol increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is known to be correlated with the incidence of cancer, in a human leukemia cell line HL-60, whereas the amount of 8-oxodG in its hydrogen peroxide (H2O2)-resistant clone HP100 was not increased. The formation of 8-oxodG in calf thymus DNA was increased by catechol in the presence of Cu2+. Catechol caused damage to 32P-labeled DNA fragments in the presence of Cu2+. When NADH was added, DNA damage was markedly enhanced and clearly observed at relatively low concentrations of catechol (<1 µM). DNA cleavage was enhanced by piperidine treatment, suggesting that catechol plus NADH caused not only deoxyribose phosphate backbone breakage but also base modification. Catechol plus NADH frequently modified thymine residues. Bathocuproine, a specific Cu+ chelator and catalase inhibited the DNA damage, indicating the participation of Cu+ and H2O2 in DNA damage. Typical hydroxyl radical scavengers did not inhibit catechol plus Cu2+-induced DNA damage, whereas methional completely inhibited it. These results suggest that reactive species derived from the reaction of H2O2 with Cu+ participates in catechol-induced DNA damage. Therefore, we conclude that oxidative DNA damage by catechol through the generation of H2O2 plays an important role in the carcinogenic process of catechol and benzene.
Abbreviations: 8-oxodG, 8-oxo-7,8-dihydro-2'-deoxyguanosine; DTPA, diethylenetriamine-N,N,N',N' ',N' '-pentaacetic acid; H2O2, hydrogen peroxide; HPLC, high-performance liquid chromatography; HPLCECD, electrochemical detector coupled to HPLC; OH·, free hydroxyl radical; O2·, superoxide anion radical; SOD, superoxide dismutase.
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Introduction
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Catechol is not only an industrial chemical but is also present in certain foods, such as onions, crude beet sugar, coffee and smoked fish. Catechol is also an important constituent of cigarette smoke. Catechol has been shown to be carcinogenic in rodents. In several experiments in rats and mice involving administration with known carcinogens, catechol strongly enhanced the incidence of papillomas of the tongue, carcinomas of the oesophagus, squamous cell carcinomas of the forestomach and adenocarcinomas of the glandular stomach (17). Recently, it was reported that catechol induced adenocarcinoma and adenomatous hyperplasia in the pyloric region of the glandular stomach of rats (812). Furthermore, catechol is a major metabolite of benzene, which is known to cause leukemia in humans and animals (1316). Catechol has been classified as a non-mutagen on the basis of the Salmonella mutagenicity test results (17). Recently, the International Agency for Research on Cancer (IARC) has classified catechol as a group 2B carcinogen (8), which is possibly carcinogenic to human. However, the mechanism of DNA damage to elicit carcinogenicity by catechol has not been clarified.
In the present study, to clarify the mechanism for DNA damage by catechol, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in a human leukemia cell line HL-60 and its hydrogen peroxide (H2O2)-resistant clone HP100 treated with catechol by using an electrochemical detector coupled to HPLC (HPLCECD). HP100 cells, which have a higher level of catalase activity than HL-60 cells (18), were used to assess whether H2O2 participates in catechol-induced DNA lesions. The oxidized nucleoside 8-oxodG has been proposed to play an important role in a number of pathological conditions, including carcinogenesis (1921). We also examined induction of apoptosis by catechol in HL-60 and HP100 cells. Furthermore, to clarify the mechanism of cellular DNA damage, we examined site-specific DNA damage by catechol using 32P 5'-end-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c-Ha-ras-1 proto-oncogene. These genes are suitable for studying the mechanisms of chemical carcinogenesis because they are known to be targets for chemical carcinogens (22,23). We also analyzed the formation of 8-oxodG in calf thymus DNA by using an HPLCECD and measured the O2· generation during the autoxidation of catechol.
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Materials and methods
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Materials
T4 polynucleotide kinase was purchased from New England Biolabs (Hitchin, UK). [
-32P]ATP (222 TBq/mmol) was from New England Nuclear (Boston, USA). Diethylenetriamine-N,N,N',N' ',N' '-pentaacetic acid (DTPA) and bathocuproinedisulfonic acid were from Dojin (Kumamoto, Japan). Superoxide dismutase (SOD, 3000 U/mg from bovine erythrocytes) and catalase (45 000 U/mg from bovine liver) were from Sigma (St Louis, MO). Catechol was from Wako Pure Chemical Industries (Osaka, Japan). 3-(Methylthio) propionaldehyde (methional) was from Tokyo Kasei (Tokyo, Japan). Copper chloride (CuCl2.2H2O) and NADH were from Nacalai Tesque (Kyoto, Japan).
Measurement of catechol-induced formation of 8-oxodG in cultured cells
HL-60 and HP100 cells were grown in RPMI 1640 supplemented with 6% FCS at 37°C under 5% CO2 in a humidified atmosphere. Cells (106 cells/ml) were incubated with catechol for 120 min at 37°C and immediately washed three times with PBS. DNA was extracted using a DNA Extractor WB kit (Wako Pure Chemical Industries). The DNA was dissolved in H2O, and treated with 8 U nuclease P1 followed by 1.2 U bacterial alkaline phosphatase. The content of 8-oxodG was determined as previously described (2426).
Detection of DNA ladder formation induced by catechol
HL-60 and HP100 cells (106 cells/ml) were treated with catechol in RPMI 1640 supplemented with 6% FCS for 4 h at 37°C under 5% CO2 in a humidified atmosphere. The media was removed and the cells were immediately washed three times with PBS. The cells were lysed and treated with RNase and proteinase K as described previously (27). The DNA was extracted with phenol/chloroform and subsequently with water-saturated ether, and precipitated with 2.5 vol ethanol. DNA was electrophoresed on a 1.4% agarose gel containing ethidium bromide in 0.5x TBE buffer.
Preparation of 32P 5'-end-labeled DNA fragments
Two fragments containing exon 1 or 2 of the human p16 tumor suppressor gene (28) were obtained by PCR amplification of human genomic DNA. Dephosphorylation with calf intestine phosphatase and phosphorylation with [
-32P]ATP and T4 polynucleotide kinase yielded a 5'-end-labeled 490 base pair (bp) fragment (EcoRI* 5841EcoRI* 6330) containing exon 1 and the 460 bp fragment (EcoRI* 9481EcoRI* 9940) containing exon 2. The 490 bp fragment was further digested with MroI to obtain the singly labeled 328 bp fragment (EcoRI* 5841MroI 6168) and the 158 bp fragment (MroI 6173EcoRI* 6330). The 460 bp fragment was also further digested with BssHII to obtain the singly labeled 309 bp fragment (EcoRI* 9481BssHII 9789) and the 147 bp fragment (BssHII 9794EcoRI* 9940). DNA fragments were also obtained from the human p53 tumor suppressor gene (29). The 32P 5'-end-labeled 650 (HindIII* 13972EcoRI* 14621) and 460 bp (HindIII* 13038EcoRI* 13507) fragments were obtained as previously described (30). The 650 bp fragment was digested with ApaI to obtain the singly labeled 211 (HindIII* 13972ApaI 14182) and 443 bp (ApaI 14179EcoRI* 14621) DNA fragments. The 460 bp fragment was digested with StyI to obtain the singly labeled 118 (HindIII* 13038StyI 13155) and 348 bp (StyI 13160EcoRI* 13507) fragments.
DNA fragments were prepared from the plasmid pbcNI, which carries a 6.6 kb BamHI chromosomal DNA segment containing the human c-Ha-ras-1 proto-oncogene. The singly labeled 261 (AvaI* 1645XbaI 1905), 341 (XbaI 1906AvaI* 2246), 98 (AvaI* 2247PstI 2344) and 337 bp (PstI 2345AvaI* 2681) fragments were obtained as previously described (31,32). For reference, nucleotide numbering starts with the BamHI site (33). An asterisk indicates 32P-labeling.
Analysis of DNA damage by catechol in the presence of Cu2+
The standard reaction mixture in a microtube (1.5 ml Eppendorf) contained catechol CuCl2, 32P-labeled DNA fragment and sonicated calf thymus DNA (10 µM/base) in 200 µl 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. After incubation at 37°C for 60 min, the DNA fragments were heated at 90°C in 1 M piperidine for 20 min and treated as previously described (31,32).
The preferred cleavage sites were determined by direct comparison of the positions of the oligonucleotides with those produced by the chemical reactions of the MaxamGilbert procedure (34) using a DNA sequencing system (LKB 2010 Macrophor). A laser densitometer (LKB 2222 UltroScan XL) was used for measurement of the relative amounts of oligonucleotides from the treated DNA fragments.
Analysis of 8-oxodG formation in calf thymus DNA by catechol in the presence of Cu2+
8-OxodG formation was determined by a modification of a reported method (35). Calf thymus DNA fragments (50 µM/base) were incubated with catechol and 20 µM CuCl2 for 60 min at 37°C. After ethanol precipitation, DNA was digested to nucleosides with nuclease P1 and calf intestine phosphatase and analyzed by HPLCECD (26).
Detection of O2· derived from catechol in the presence of Cu2+
The amount of O2· generation was determined by the measurement of cytochrome c reduction. The mixture containing 40 µM ferricytochrome c, 100 µM catechol, 20 µM CuCl2 and 2.5 µM DTPA in 1 ml 10 mM sodium phosphate buffer (pH 7.8) was incubated at 37°C. A maximum absorption at 550 nm due to ferrocytochrome c formed by ferricytochrome c reduction was measured with a UV-visible recording spectrophotometer. The content of O2· at a low estimate was calculated by subtracting absorbance with SOD from that without SOD at 550 nm (
= 21.1x103 M1. cm1).
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Results
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Formation of 8-oxodG in human cultured cells by catechol
To investigate the induction of cellular oxidative DNA damage by catechol, we measured the content of 8-oxodG, a relevant indicator of oxidative base damage, in the DNA of HL-60 and HP100 cells treated with catechol. Catechol treatment resulted in increased 8-oxodG content in DNA extracted from treated HL-60 cells in a dose-dependent manner (Figure 1
). The content of 8-oxodG of DNA in HL-60 cells treated with 20 µM catechol was significantly increased in comparison with non-treated cells. However, catechol did not significantly increase the amount of 8-oxodG in HP100 cells (Figure 1
). It was reported that HP100 cells are ~340-fold more resistant to H2O2 than the parent cells, HL-60 (18). Therefore, given this result, it suggests that generation of H2O2 plays an important role in catechol-induced 8-oxodG formation.

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Fig. 1. Contents of 8-oxodG in DNA of HL-60 and HP100 cells treated with catechol. HL-60 () and HP100 ( ) cells (4.0x106 cells) were incubated with various concentrations of catechol for 120 min at 37°C and the DNA was extracted immediately. The extracted DNA was subjected to enzyme digestion and analyzed by HPLCECD as described in Materials and methods. Results are expressed as mean ± SE of values obtained from six independent experiments. The asterisk indicates a significant difference when compared with control by t-test (P < 0.01).
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Detection of DNA ladder formation induced by catechol
Figure 2
shows DNA ladder formation, which is associated with apoptosis, in cells treated with catechol. DNA ladder formation was just visible at 20 µM and greatly increased at 50 µM catechol in HL-60 cells. However, catechol treatment did not lead to DNA ladder formation in HP100 cells. These results also suggest that generation of H2O2 plays an important role in catechol-mediated apoptosis.

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Fig. 2. Detection of DNA ladder formation in cultured cells treated with catechol. HL-60 and HP100 cells (1.0x106 cells) were treated with catechol for 4 h at 37°C. The cells were lysed and DNA was extracted and analyzed by conventional electrophoresis as described in Materials and methods. Marker lane, size marker DNA ( X174/HaeIII digest).
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Damage to 32P-labeled DNA fragments induced by catechol
Figure 3
shows an autoradiogram of DNA damage induced by catechol. Catechol caused DNA damage depending on its concentration in the presence of Cu2+. Even without piperidine treatment, oligonucleotides were formed by catechol in the presence of Cu2+, indicating breakage of the deoxyribose phosphate backbone. The amount of oligonucleotides was increased by piperidine treatment. Since an altered base is readily removed from its sugar by the piperidine treatment, this suggests that base modification was induced by catechol in the presence of Cu2+. Catechol did not cause DNA damage in the presence of other metal ions (Fe3+, Co2+, Ni2+, Mn2+ or Mg2+) (data not shown).

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Fig. 3. Autoradiogram of 32P-labeled DNA fragment incubated with catechol and NADH in the presence of Cu2+. Reaction mixtures contained 32P 5'-end-labeled 211 bp fragment, 10 µM/base calf thymus DNA, the indicated concentrations of catechol, 100 µM NADH and 20 µM CuCl2 in 10 mM phosphate buffer (pH 7.8) containing 5 µM DTPA. After the incubation at 37°C for 60 min, followed by piperidine treatment, the treated DNA fragments were electrophoresed on an 8% polyacrylamide, 8 M urea gel (12x16 cm) and the autoradiogram was obtained by exposing X-ray film to the gel.
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DNA damage was significantly enhanced by the addition of 100 µM NADH. NADH is a reductant existing at high concentrations (100200 µM) in the cell (36). The magnitude of DNA damage induced by 1 µM catechol in the presence of NADH was greater than that of 20 µM catechol without NADH.
Effects of free hydroxyl radical (OH·) scavengers, catalase and bathocuproine on DNA damage
Figure 4
shows the effect of OH· scavengers, catalase and bathocuproine, on DNA damage induced by catechol and NADH in the presence of Cu2+. Typical OH· scavengers, ethanol, mannitol, sodium formate and DMSO, showed no or little inhibitory effect on DNA damage, whereas methional did inhibit DNA damage. Catalase and bathocuproine, a Cu+-specific chelator, inhibited DNA damage completely. These results suggest that both Cu+ and H2O2 play important roles in DNA damage. SOD did not inhibit DNA damage by catechol and NADH in the presence of Cu2+.

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Fig. 4. Effects of scavengers on DNA damage induced by catechol and NADH in the presence of Cu2+. The reaction mixture contained 32P 5'-end-labeled 211 bp fragment, 10 µM/base sonicated calf thymus DNA, 2 µM catechol, 100 µM NADH, 20 µM CuCl2 and scavenger in 200 µl 10 mM sodium phosphate buffer at pH 7.8 containing 5 µM DTPA. Scavenger was added where indicated. After incubation at 37°C for 60 min, followed by piperidine treatment, DNA fragments were analyzed by the method described in Figure 3 . The concentration of scavengers was as follows: 0.8 M ethanol, 0.2 M mannitol, 0.2 M sodium formate, 0.8 M DMSO, 1.0 M methional, 50 U catalase and 50 U SOD.
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Site specificity of DNA damage by catechol
Patterns of DNA cleavage induced by catechol in the presence of Cu2+ were determined by DNA sequencing using the MaxamGilbert procedure (34). The relative intensity of DNA cleavage obtained by scanning autoradiogram with a laser densitometer is shown in Figure 5
. Catechol frequently induced piperidine-labile sites at thymine residues, especially at the 5'-GTC-3' sequence in DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene (Figure 5A
) and p53 and p16 tumor suppressor genes (data not shown). A similar DNA cleavage pattern was observed with and without NADH in the same system (Figure 5B
). When denatured DNA was used, damage occurred more frequently at guanine residues (data not shown). Previously, we demonstrated that in the presence of Cu2+, NADH plus H2O2 frequently induced piperidine-labile sites at thymine residues (3739). This site specificity was almost the same as that of catechol.

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Fig. 5. Site specificity of DNA cleavage induced by catechol and NADH in the presence of Cu2+. Reaction mixtures containing 32P 5'-end labeled 341 bp DNA fragment, 10 µM/base calf thymus DNA, 20 µM CuCl2 and (A) 20 µM catechol or (B) 1 µM catechol and 100 µM NADH in 10 mM phosphate buffer (pH 7.8) containing 5 µM DTPA were incubated at 37°C for 60 min. After piperidine treatment, DNA fragment was electrophoresed on an 8% polyacrylamide/8 M urea gel and the autoradiogram was obtained by exposing X-ray film to the gel.
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Formation of 8-oxodG in calf thymus DNA by catechol and NADH in the presence of Cu2+
Catechol increased 8-oxodG content in calf thymus DNA in the presence of Cu2+ (Figure 6
). The amount of 8-oxodG increased with increasing catechol concentration. The addition of NADH enhanced catechol plus Cu(II)-induced 8-oxodG formation. Catechol did not cause 8-oxodG formation in the absence of Cu2+.

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Fig. 6. Formation of 8-oxodG induced by catechol and Cu2+ in the presence and absence of NADH. Calf thymus DNA (100 µM/base) was incubated with catechol, 100 µM NADH and 20 µM CuCl2 in 4 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA at 37°C for 60 min. After ethanol precipitation, the DNA was subjected to enzyme digestion and analyzed by HPLCECD as described in Materials and methods.
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O2· generation from the reaction of catechol with Cu2+
Generation of O2· was investigated by cytochrome c reduction (Figure 7
). In the catechol and Cu2+ system, cytochrome c was actually reduced and the reduction was inhibited in the presence of SOD due to the conversion of O2· into H2O2. The amount of O2· generation was estimated from the difference in cytochrome c reduction with and without SOD. The amount of O2· was increased within 4 min, indicating the O2· was generated in a relatively short time period compared with DNA damage.

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Fig. 7. Time course of generation of O2· during catechol and Cu2+ treatment in the presence and absence of SOD. Reaction mixtures contained 40 µM cytochrome c and 100 µM catechol in the presence of 100 U/ml SOD in 10 mM phosphate buffer (pH 7.8) containing 2.5 µM DTPA. A maximum absorption at 550 nm was measured at 37°C with a UV-visible spectrophotometer every 1 min for 4 min.
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Discussion
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In this study, we demonstrated that the content of 8-oxodG in HL-60 cells was increased by catechol, whereas the content of 8-oxodG in HP100 cells was not increased. DNA base damage in the form of 8-oxodG is a prominent indicator of oxidative stress and has been well-characterized as a premutagenic lesion in mammalian cells (1921). Catalase activity of HP100 cells was 18-fold higher than that of HL-60 cells (18). These results suggest that catechol is capable of causing oxidative DNA damage in human cultured cells, and that generation of H2O2 plays a critical role in catechol-mediated DNA damage. Numerous studies have indicated that the formation of 8-oxodG causes misreplication of DNA that may lead to mutation or cancer (21,40). Thus, oxidative DNA damage seems to be relevant to the carcinogenic process of catechol.
To clarify the mechanism of cellular DNA damage, we investigated DNA damage by catechol using 32P-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c-Ha-ras-1 proto-oncogene. Catechol induced DNA damage in the presence of Cu2+. The effect of piperidine treatment indicates that catechol induced not only breakage of the deoxyribose phosphate backbone but also base alteration. The formation of 8-oxodG in calf thymus DNA by catechol was observed in the presence of Cu2+. The oxidative DNA damage was markedly enhanced by the addition of NADH. Several studies indicate that NADH may react non-enzymatically with some quinones and mediate their reduction (4144). These results suggest the generation of catechol from its oxidized products through the reduction by NADH. The biological importance of NADH as a nuclear reductant has been described (45). The concentration of NADH in certain tissues was estimated to be as high as 100200 µM (36). Thus, the NADH-dependent redox cycle of catechol may continuously generate reactive oxygen species and mediate oxidative DNA damage in cells.
In order to clarify what kind of reactive oxygen species cause oxidative DNA damage, the effects of various scavengers and site specificity of DNA damage were examined. Catechol plus NADH frequently induced piperidine-labile sites at thymine residues with the sequence 5'-GTC-3' in the presence of Cu2+. This result supports the involvement of reactive species other than OH·, as DNA damage caused by OH· shows little site specificity (37,46). Furthermore, typical OH· scavengers showed little or no inhibitory effect on DNA damage, whereas DNA damage was inhibited by methional. Methional scavenges not only OH· but also a variety of reactive species other than OH· (47). The inhibitory effects of bathocuproine and catalase on Cu2+-mediated DNA damage indicate that Cu+ and H2O2 have important roles in the production of the active species responsible for causing DNA damage.
On the basis of these results, a possible mechanism of oxidative DNA damage by catechol and NADH in the presence of Cu2+ has been proposed in Figure 8
. It is reasonable to speculate that catechol undergoes Cu2+-mediated autoxidation to generate Cu+ and semiquinone radical. Cu+ reacts with O2 to generate O2· and subsequently H2O2. Formed Cu+ bound to DNA interacts with H2O2, resulting in the formation of a Cu(I)hydroperoxo complex such as DNACu(I)OOH (48). Furthermore, the reactive oxygen species can be produced abundantly through the reduction of the oxidized form, such as semiquinone radical or 1,2-benzoquinone, by NADH non-enzymatically. The Cu(I)hydroperoxo complex may be considered to be a bound hydroxyl radical, which can release OH· causing DNA damage. The OH· released from a bound hydroxyl radical immediately attacks an adjacent constituent of DNA before it can be scavenged by OH· scavengers (49). In addition, catechol-type compounds, such as carcinogenic catechol estrogens and flavanoids, also induced oxidative DNA damage through H2O2 generation (5052).
The binding of copper to DNA and/or protein in chromatin is proposed to serve physiological functions (53), whereas copper bound to DNA and/or protein may provide an adventitious site for deleterious redox reactions (54). Copper ions bind to non-histone proteins and cause much stronger ascorbate-mediated DNA damage than iron (55). Several studies have indicated that copper has the ability to catalyze the production of reactive oxygen species and to mediate oxidative DNA damage (37,38,49,56,57). The present work suggests that copper is an important factor in oxidative DNA damage by catechol and NADH.
It is well-known that catechol has strong promotion activity. Many investigations have indicated that catechol strongly enhances cancer development in rats and mice initiated with carcinogens, such as benzo[a]pyrene and N-methyl-N'-nitro-N-nitrosoguanidine (17). Recent observations have suggested that some tumor promoters act to produce DNA damage mediated by reactive oxygen species (5862). In this study, we demonstrated that catechol could induce metal-dependent H2O2 generation and subsequent damage to DNA fragments obtained from the human p53 and p16 tumor suppressor genes. It is speculated that the genetic damage might lead to inactivation of these tumor suppressor genes, which is consistent with the step of tumor promotion in a multistage process of carcinogenesis.
The relationship between DNA damage, apoptosis and carcinogenesis has attracted considerable interest. The present study demonstrated that DNA ladder formation, which is associated with apoptosis, by 50 µM catechol was observed in HL-60 cells, whereas in HP100 cells, no DNA ladders were observed. In addition, the content of 8-oxodG of DNA in HL-60 cells treated with 20 µM catechol was significantly increased in comparison with non-treated cells. These results suggest that catechol treatment generated H2O2 to induce 8-oxodG formation, preceding apoptosis. Our previous studies have shown that oxidative stress such as UVA irradiation induced 8-oxodG formation, followed by the loss of mitochondrial membrane potential (
m) and subsequent activation of caspase-3, resulting in apoptosis (25,63). These results have led us to propose two possible fates for the cells with DNA damage; one is apoptosis, and the other is mutation leading to carcinogenesis. The cells that incur strong DNA damage and undergo apoptosis are no longer candidates for cancer cells. When weak DNA damage was induced, the cellular response allows the repair of damage. However, if the damage failed to be repaired, mutagenic lesions could be propagated and might lead to carcinogenesis.
Catechol is a ubiquitous natural and industrial glandular stomach carcinogen and tumor promoter, which humans ingest or are exposed to through foods, coffee, cigarette smoke and oxidative type of hair dyes. Furthermore, benzene is converted to catechol by cytochrome P-450. The presence of catechol in human urine has been reported previously (64). Catechol concentrations in the urine of non-smokers on unrestricted diets and workers exposed to catechol were 10 ± 11.7 (about 70 µM) and 24.2 ± 11.7 mg/day (mean ± SD) (65). In addition, levels of catechol in urine from benzene exposed workers were significantly higher than that in unexposed subjects (66). We demonstrated that 20 µM catechol caused oxidative DNA damage in cells. Furthermore, in the presence of NADH, a low concentration (0.5 µM) of catechol induced metal-mediated DNA damage. Therefore, it is concluded that NADH and Cu2+-mediated oxidative DNA damage by catechol plays an important role in the carcinogenic process caused by catechol and benzene. Further investigation of catechol metabolism and toxicity are also needed in view of public health.
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Notes
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3 To whom correspondence should be addressed Email: kawanisi{at}doc.medic.mie-u.ac.jp 
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Acknowledgments
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This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.
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Received December 31, 2000;
revised March 26, 2001;
accepted April 11, 2001.