Oxidation of 5'-site guanine at GG and GGG sequences induced by a metabolite of carcinogenic heterocyclic amine PhIP in the presence of Cu(II) and NADH

Mariko Murata and Shosuke Kawanishi,1

Department of Hygiene, Mie University School of Medicine, Tsu, Mie, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Adduct formation has been considered to be a major causal factor of DNA damage by carcinogenic heterocyclic amines. By means of experiments with an electrochemical detector coupled to a high-performance liquid chromatograph, we revealed that N-hydroxy metabolite of 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) induced the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in the presence of Cu(II). Addition of an endogenous reductant NADH enhanced the 8-OH-dG formation. Experiments with 32P-labeled DNA fragments showed that this metabolite [PhIP(NHOH)] caused 8-hydroxylation of guanines in the presence of Cu(II) and NADH, and subsequent treatment with formamidopyrimidine-DNA glycosylase led to chain cleavages at the 5'-site guanine of GG and GGG sequences. Interestingly, antioxidant enzyme SOD enhanced the intensity of DNA damage, and thymine residues were appended to its guanine-predominant cleavage sites. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited the DNA damage, suggesting the involvement of H2O2 and Cu(I). A UV-visible spectroscopic study indicated that Cu(II) and SOD catalyze the autoxidation of PhIP(NHOH). These results suggest that Cu(II)-dependent autooxidation of PhIP(NHOH) coupled with NADHmediated reduction of its oxidized product form redox cycle, resulting in oxidative DNA damage by low concentrations of PhIP(NHOH). We conclude that in addition to DNA adduct formation, oxidative DNA damage may be involved in the carcinogenic process of PhIP.

Abbreviations: Fpg, formamidopyrimidine-DNA glycosylase;; H2O2, hydrogen peroxide;; HPLC, high-performance liquid chromatography;; HPLC-ECD, HPLC equipped with an electrochemical detector;; O2–, superoxide anion radical;; OH, free hydroxyl radical;; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine (also known as 8-oxodG, 8-oxo-7,8-dihydro-2'-deoxyguanosine);; PhIP (NHOH), 2-hydroxyamino-1-methyl-6-phenylimidazo [4,5-b] pyridine;; PhIP(NO), 2-nitroso-1-methyl-6-phenylimidazo [4,5-b] pyridine;; PhIP(NO2), 2-nitro-1-methyl-6-phenylimidazo [4,5-b] pyridine; DTPA, diethylenetriamine-N,N,N',N'',N''-pentaacetic acid;; PhIP, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine;; SOD, superoxide dismutase.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The heterocyclic amines are a family of mutagenic/carcinogenic compounds produced during the pyrolysis of creatine, amino acids and proteins. (1–5). 2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) is one such heterocyclic amine isolated from cooked beef, chicken, fish and pork (1–5). In investigations of foods for the presence of multiple heterocyclic amines, PhIP is usually found to be the most abundant. In addition, PhIP has also been found in mainstream cigarette smoke condensate (1). Oral administration of PhIP produces lymphomas in mice (6), adenocarcinomas of the small and large intestine, and mammary adenocarcinomas in rats (7). Thus, PhIP has been estimated to possess possible carcinogenic risk to humans (Group 2B) by the International Agency for Research on Cancer (1).

Heterocyclic amines are metabolically activated to DNA-binding products via the exocyclic amino group. PhIP is oxidized to a N-hydroxy derivative [PhIP(NHOH)] in the liver by cytochrome P450 enzymes, and the latter is esterified by acetyltransferases or sulfotransferases to its ultimate carcinogen (1–5,8–10). The adduct formation is thought to be crucially important and PhIP-DNA adducts have been detected in human tissues (5). However, there are several reports that indicate no straightforward relationship between PhIP-DNA adduct formation and carcinogenicity (11–13). Recently, El-Bayoumy et al (14) reported that oral administration of PhIP to rats increased the 8-hydroxy-2'-deoxyguanosine (8-OH-dG) level in mammary gland. Furthermore, it has been reported that several antioxidants significantly inhibited PhIP-induced mutagenicity (15) and carcinogenicity (13). These reports lead us to consider that reactive oxygen species may participate in the heterocyclic amine-induced tumor development. Hayatsu and his colleagues (16,17) showed that the N-hydroxy derivative of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) produces intracellular reactive oxygen species that can damage DNA in mouse cells in culture. We reported that several N-hydroxy derivatives of aromatic amines can cause oxidative DNA damage (18–20). Therefore, DNA adducts themselves may not be sufficient for the expression of carcinogenicity, although DNA adduct formation is a prerequisite. There remains a possibility that oxidative DNA damage also plays a role in carcinogenesis induced by PhIP.

In this study, we investigated whether PhIP(NHOH) can cause oxidative DNA damage or not, using 32P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. We analyzed 8-OH-dG formation in calf thymus DNA by PhIP(NHOH) in the presence of Cu(II) and NADH. Furthermore, in order to clarify the mechanism of oxidative DNA damage, spectral changes during the autoxidation of PhIP(NHOH) were measured using a UV-visible spectroscopy.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Restriction enzymes (ApaI, AvaI, EcoRI, HindIII, XbaI, PstI) and T4 polynucleotide kinase were purchased from New England Biolabs. Calf intestine phosphatase was from Roche Molecular Biochemicals (Mannheim, Germany). [{gamma}-32P]ATP (222 TBq/mmol) was from New England Nuclear. PhIP(NHOH) was supplied by NCI Chemical Carcinogen Reference Standard Repository in Midwest Research Institute (Menlo Park, CA). ß-Nicotinamide adenine dinucleotide disodium salt (reduced form) (NADH) was purchased from Kohjin (Tokyo, Japan). Diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA) and bathocuproinedisulfonic acid were from Dojin Chemicals (Kumamoto, Japan). Acrylamide, bisacrylamide, and piperidine were from Wako Chemicals (Osaka, Japan). CuCl2, D-mannitol and ethanol were from Nacalai Tesque (Kyoto, Japan). Calf thymus DNA, CuZn-SOD (3000 units/mg from bovine erythrocytes), methional and catalase (45 000 units/mg from bovine liver) were from Sigma Chemical (St Louis, MO). Nuclease P1 was from Yamasa Shoyu (Chiba, Japan). Formamidopyrimidine-DNA glycosylase (Fpg, 20 000 units/mg from Escherichia coli) was from Trevigen (Gaithersburg, MD). Bicarbonate buffer (pH 7) was freshly prepared each time, and pH adjustment (range; pH 7.2–7.8) was done by bubbling of carbon dioxide gas.

Preparation of 32P-5'-end-labeled DNA fragments
DNA fragments were obtained from the human p53 tumor suppressor gene (21) and the c-Ha-ras-1 protooncogene (22). The DNA fragment of the p53 tumor suppressor gene was prepared from pUC18 plasmid. The singly 32P-5'-end-labeled 211 bp fragment (HindIII* 13972–ApaI 14182) was obtained according to the method described previously (23). DNA fragments were also prepared from plasmid pbcNI, which carries a 6.6 kb BamHI chromosomal DNA segment containing the human c-Ha-ras-1 protooncogene (24). The singly labeled 261 bp fragment (Ava I* 1645–Xba I 1905) and 337 bp fragment (Pst I 2345–Ava I* 2681) were obtained according to the method described previously (24). The asterisk indicates 32P-labeling.

Detection of damage to isolated DNA
The standard reaction mixture in a 1.5 ml microtube contained indicated concentrations of PhIP(NHOH), NADH, CuCl2, 32P-5'-end-labeled DNA fragment and calf thymus DNA in 200 µl of 10 mM bicarbonate buffer (pH 7) containing 2.5 µM DTPA. After incubation at 37°C for the indicated time, the DNA fragments were heated at 90°C in 1 M piperidine for 20 min where indicated and treated as described previously (24). In certain experiments, the DNA was treated with six units of Fpg protein in 10 µl of the reaction buffer (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10 mM EDTA and 0.1 mg/ml BSA) at 37°C for 2 h. Fpg protein catalyzes the excision of 8-OH-dG and other oxidized bases such as guanidinohydantoin and spiroiminodihydantoin, which are resulting from oxidation of 8-OH-dG (25,26). The preferred cleavage sites were determined by direct comparison of the positions of the oligonucleotides with those produced by the chemical reactions of the Maxam-Gilbert procedure (27) using a DNA-sequencing system (LKB 2010 Macrophor). A laser densitometer (LKB 2222 UltroScan XL) was used for the measurement of the relative amounts of oligonucleotides from treated DNA fragments.

Measurement of 8-OH-dG formation in calf thymus DNA
Native or denatured DNA fragments (100 µM per base) from calf thymus were incubated with PhIP(NHOH), NADH and CuCl2 for the indicated duration at 37°C. Denatured DNA fragments were obtained by heating at 90°C for 5 min followed by chilling on ice before incubation. After ethanol precipitation, DNA was enzymatically digested to the nucleosides and analyzed by high-pressure liquid chromatograph with an electrochemical detector (HPLC-ECD), as described previously (28).

UV-visible spectra measurements
UV-visible spectra were measured with a UV-visible spectrometer (UV-2500PC, Shimadzu, Kyoto). The reaction mixture contained PhIP(NHOH) in 10 mM bicarbonate buffer (pH 7) containing 2.5 µM DTPA. Where indicated, CuCl2 or SOD was added to the reaction mixtures. The spectra of the mixtures were measured repeatedly at 37°C for the indicated duration. The rate of PhIP(NHOH) autoxidation was calculated by the decrease of absorbance at 315 nm (1.70 x 103 M–1cm–1) during the first 5 min. To analyze the redox reaction with 100 µM NADH, the change of absorption at 340 nm was measured with a UV-visible spectrometer. The decrease in absorbance of NADH at 340 nm ({varepsilon} = 6.22 x 103 M–1cm–1) is due to its oxidation to NAD+.


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Damage to 32P-labeled DNA fragments by PhIP (NHOH) in the presence of NADH and Cu(II)
Figure 1Go shows an autoradiogram of DNA fragments treated with PhIP(NHOH) in the presence and absence of NADH and Cu(II). Oligonucleotides were detected on the autoradiogram as a result of DNA cleavage. In the absence of PhIP(NHOH), DNA damage was not observed with NADH and Cu(II) under the conditions used. PhIP(NHOH) alone and PhIP(NHOH) plus NADH did not cause DNA damage. PhIP(NHOH) could induce Cu(II)-mediated DNA damage without NADH, although much higher concentrations of PHIP(NHOH), above 50 µM, were required under the condition used (data not shown). The intensity of DNA damage increased with increasing concentration of PhIP(NHOH) (Figure 1Go) and incubation time (data not shown) in the presence of Cu(II) and NADH. The increase of oligonucleotides by piperidine treatment suggested that not only strand breakage but also base modification and/or liberation were induced. When denatured DNA was used, oligonucleotides increased (data not shown). Regarding DNA adduct formation, it is known that covalent binding to DNA, at least to the N7 position of guanine, can be detected by piperidine treatment (29,30). When Cu(II) was not added, PhIP(NHOH) did not increase formation of oligonucleotides with piperidine treatment. Therefore, there is no or little DNA adduct formation under the condition used, although there is a possibility that some DNA adducts might be underrepresented dependent on its resistance to piperidine.



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Fig. 1. DNA damage by PhIP(NHOH) in the presence of NADH and Cu(II). The reaction mixture containing the 32P-5'-end-labeled 337 bp DNA fragment, 10 µM/base of calf thymus DNA, 20 µM CuCl2, 200 µM NADH, and the indicated concentrations of PhIP(NHOH) was incubated at 37°C for 60 min, and followed by piperidine treatment.

 
Effects of scavengers and bathocuproine on DNA damage induced by PhIP(NHOH) in the presence of Cu(II) and NADH
Figure 2Go shows the effects of scavengers and bathocuproine, a Cu(I)-specific chelator, on DNA damage induced by PhIP(NHOH) in the presence of Cu(II) and NADH. Inhibition of DNA damage by catalase and bathocuproine suggests the involvement of hydrogen peroxide (H2O2) and Cu(I). Methional inhibited the DNA damage, although other typical hydroxyl radical (oOH) scavengers such as ethanol, mannitol and sodium formate, did not. Interestingly, SOD enhanced DNA damage induced by PhIP(NHOH) in the presence of Cu(II) and NADH.



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Fig. 2. Effects of scavengers and bathocuproine on DNA damage by PhIP(NHOH) in the presence of NADH and Cu(II). The reaction mixture containing the 32P-5'-end-labeled 211 bp DNA fragment, 10 µM/base of calf thymus DNA, 20 µM CuCl2, 200 µM NADH, 0.5 µM PhIP(NHOH) and a scavenger (5 % (v/v) ethanol; 0.1 M mannitol; 0.1 M sodium formate; 0.1 M methional; 30 units of SOD; 30 units of catalase; 50 µM bathocuproine) was incubated at 37°C for 30 min, and then the DNA was treated with hot piperidine.

 
Site specificity of DNA cleavage by PhIP(NHOH) in the presence of NADH and Cu(II)
An autoradiogram was obtained and scanned with a laser densitometer to measure relative intensity of DNA cleavage in the human c-Ha-ras-1 protooncogene and the p53 tumor suppressor gene as shown in Figures 3–4GoGo. PhIP(NHOH) caused oxidation of guanines in the presence of Cu(II) and NADH, and subsequent treatment with formamidopyrimidine-DNA glycosylase led to chain cleavages at the 5'-site guanine of GG and GGG sequences (Figure 3Go). It is noteworthy that the damaged guanine residue of the CGG and AGG sequences are codons 248 and 249, which are well-known mutational hotspots (31–33) of the p53 gene (Figure 3AGo). PhIP(NHOH) induced piperidine-labile sites preferentially at the 5'-site guanine of GG and GGG sequences in the presence of Cu(II) and NADH (Figure 4AGo). When SOD was added, thymine residues were appended to its guanine-predominant cleavage sites (Figure 4BGo). Similar DNA cleavage patterns were observed in the human p53 tumor suppressor gene (data not shown).



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Fig. 3. Site specificity of DNA cleavage induced by PhIP(NHOH) in the presence of NADH and Cu(II), followed by Fpg treatment. The reaction mixture containing the 32P-5'-end-labeled 211 bp DNA fragment (A) and 261 bp DNA fragment (B), 10 µM/base of calf thymus DNA, 20 µM CuCl2, 200 µM NADH, 1 µM PhIP(NHOH) was incubated at 37°C for 60 min, and then DNA was treated with Fpg protein.

 


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Fig. 4. Site specificity of DNA cleavage induced by PhIP(NHOH) in the presence of NADH and Cu(II), and the effect of SOD on it. The reaction mixture containing the 32P-5'-end-labeled 337 bp DNA fragment, 10 µM/base of calf thymus DNA, 20 µM CuCl2, 200 µM NADH, 0.5 µM (A) or 0.2 µM (B) PhIP(NHOH) in the presence (B) and absence (A) of 30 units SOD was incubated at 37°C for 30 min, and then the DNA was treated with hot piperidine.

 
Formation of 8-OH-dG in calf thymus DNA by PhIP(NHOH) in the presence of NADH and Cu(II)
Using HPLC-ECD, we analyzed the Cu(II)-mediated 8-OH-dG formation in calf thymus DNA treated with PhIP(NHOH) in the presence and absence of NADH (Figure 5Go). The amount of 8-OH-dG increased with the concentration of PhIP(NHOH), and the addition of NADH enhanced the formation ~20-fold. The formation of 8-OH-dG increased ~5-fold after DNA denaturation (data not shown).



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Fig. 5. Formation of 8-OH-dG induced by PhIP(NHOH) in the presence of NADH and Cu(II). The reaction mixture containing calf thymus DNA (100 µM/base), the indicated concentrations of PhIP(NHOH), 20 µM CuCl2 in the presence (squares) and absence (circles) of 200 µM NADH was incubated at 37°C for 60 min, and then the DNA was enzymatically digested into nucleosides, and 8-OH-dG formation was measured with an HPLC–ECD.

 
UV-visible spectroscopic study on the autoxidation of PhIP(NHOH)
The UV-visible spectra of PhIP(NHOH) changed only a little during 30 min, suggesting very slow autoxidation in the absence of Cu(II). PhIP(NHOH) has the absorbance maximum at 270 nm and 315 nm. When Cu(II) was added, PhIP(NHOH) showed a rapid decrease in the absorbance maximum at 270 nm and 315 nm with increasing the absorbance maximum at 384 nm during 30 min (Figure 6AGo). Figure 6BGo showed the initial rate in decomposition of PhIP(NHOH) in the presence and absence of Cu(II) and SOD assessed by the decrease of absorbance at 315 nm during the first 5 min. Addition of SOD and/or Cu(II) accelerated the initial velocity of PhIP(NHOH) autoxidation.



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Fig. 6. UV-spectral changes of PhIP(NHOH) and NADH consumption in the presence of Cu(II). (A) The reaction mixture containing 50 µM PhIP(NHOH) and 5 µM CuCl2 was kept at 37°C. The spectral tracing was initiated by addition of CuCl2 and repeated every 5 min for 30 min. (B) Effect of Cu(II) and/or SOD on the initial rate of decomposition of PhIP(NHOH). Control; 50 µM PhIP(NHOH), SOD(+); 50 µM PhIP(NHOH) + 150 units/ml SOD, Cu(II)(+); 50 µM PhIP(NHOH) + 5 µM CuCl2, SOD(+) + Cu(II)(+); 50 µM PhIP(NHOH) + 150 units/ml SOD + 5 µM CuCl2. (C) The reaction mixture containing the indicated concentrations of PhIP(NHOH), 100 µM NADH and 5 µM Cu(II) was measured every 5 min for 30 min.

 
The spectral changes of NADH were measured to clarify the effect of NADH oxidation on the reaction mixture of PhIP(NHOH) and Cu(II). Figure 6CGo shows that the Cu(II)-mediated redox reaction of PhIP(NHOH) with 100 µM NADH was observed by the decreasing absorbance at 340 nm of NADH (reduced form) for 30 min. The higher concentration of PhIP(NHOH) oxidized the greater amount of NADH. When Cu(II) was omitted, NADH oxidation occurred only a little.


    Discussion
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 Abstract
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 Materials and methods
 Results
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The present study has demonstrated that the N-hydroxy derivative of a heterocyclic amine PhIP has the ability to cause oxidation of the 5'-site guanine of GG and GGG sequences in the presence of Cu(II) and NADH. The existence of NADH made PhIP(NHOH)-induced DNA damage more efficient, >20-fold, although high concentrations of PhIP(NHOH) could cause Cu(II)-mediated DNA damage. PhIP(NHOH) could induce Cu(II)/NADH-dependent DNA damage at the 5'-site guanine of GG and GGG sequences. Addition of SOD enhanced the DNA damage and altered the site specificity. The UV-visible spectroscopic study showed that Cu(II) mediated autoxidation of PhIP(NHOH) to the oxidized form. NADH was consumed in the reaction mixture of PhIP(NHOH) and Cu(II).

Comet assay revealed that milk-activated PhIP induced DNA damage in breast epithelial cells (34). The present study also indicated that PhIP(NHOH) induced DNA damage in cell-free system. Since our data were obtained from a simplified in vitro model system, the amount of DNA damage cannot be directly extrapolated to biological system. However, DNA damage can be detected sensitively using the putative ultimate carcinogens and the results will provide the mechanistic consideration.

The hypothetical mechanism of oxidative DNA damage by PhIP(NHOH) shown in Figure 7Go can account for most of the observations. PhIP(NHOH) is oxidized into the hydronitroxide radical in the presence of Cu(II), followed by the autoxidation to probably 2-nitroso-1-methyl-6-phenylimidazo [4,5-b] pyridine [PhIP(NO)] but not to 2-nitro-1-methyl-6-phenylimidazo [4,5-b] pyridine [PhIP(NO2)]. PhIP(NO2) has the absorbance maximum at 350 nm (35), and therefore, the absorbance maximum at 384 nm observed in Figure 6AGo may be attributed to PhIP(NO). Relevantly, we observed that several N-hydroxy derivatives of aromatic amines were autoxidized to nitroso derivatives (18,36). On the basis of these results and the literature, it is speculated that Cu(II) and SOD catalyzes PhIP(NHOH) autoxidation to PhIP(NO). PhIP(NO) is reduced to PHIP(NHOH) by NADH, and NADH itself is oxidized to NAD, and further to NAD+ with formation of O2–. During the autoxidation of PhIP(NHOH), DNA-bound Cu(II) can undergo reduction to Cu(I), resulting in formation of Cu(II)/Cu(I) redox cycling. O2– is dismutated to H2O2 to form the DNA-Cu(I)-H2O2 complex (24). Relevantly, it was reported that H2O2 reacts with Cu(I) to form reactive species capable of causing DNA damage (24,37,38). Methional completely inhibited the DNA damage, whereas typical OH scavengers did not. Methional scavenges not only the OH, but it can also scavenge crypto-OH radicals (39,40). Therefore, it is reasonably considered that a short-lived ternary complex, such as DNA-Cu(I)-OOH, is then formed by the reaction of DNA-bound Cu(I) with H2O2 and that the electron is then transferred to the peroxide, forming OH, which reacts immediately with the DNA at the copper binding site before being scavenged by OH scavengers.



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Fig. 7. A proposed mechanism of oxidative DNA damage induced by PhIP(NHOH) in the presence of NADH and Cu(II).

 
Endogenous reductants, such as NADH, can reduce nitroso derivatives to N-hydroxy derivatives with the generation of O2 from O2, forming a redox cycle (41–43). Very powerful one-electron oxidants would be able to oxidize NADH via a mechanistic pathway involving one-electron transfer (44). Several studies indicate that NADH may react nonenzymatically with some xenobiotics and mediate their reduction (45,46). The cycling of redox reactions would cause the DNA damage with excessive generation of reactive oxygen species by the low concentrations of PhIP(NHOH).

The antioxidant enzyme SOD enhanced the DNA damage instead of defense against oxidative stress. Several papers have reported that SOD enhanced oxidative DNA damage (47–50). The enhancing effect is probably due to SOD-catalyzed consumption of O2–, resulting in acceleration of PhIP(NHOH) autoxidation as confirmed by UV-visible spectrometer. However, the alteration of site specificity of DNA damage observed here cannot be explained by the acceleration of autooxidation. A recent report (51) explained the SOD-related alteration of site specificity of DNA damage induced by Cu(II) and H2O2 as follows; SOD formed Cu(I) in the presence of Cu(II) and H2O2, and the excess Cu(I) may induce the proton transfer from guanine to cytosine, and from adenine to thymine residues, resulting in the alteration. Although the mechanism for the alteration of its site specificity by SOD is not completely clarified, the SOD effect is noteworthy in relation to the carcinogenicity of PhIP.

This study demonstrated that PhIP(NHOH) could induce Cu(II)/NADH-dependent DNA damage at the 5'-site guanine of GG and GGG sequences. In addition, PhIP(NHOH) caused cleavage at the guanine residues of the 5'-CGG-3' sequence (codon 248) and 5'-AGG-3' sequence (codon 249), which are known hotspots of the p53 gene (31–33). Several studies revealed that colon cancers of rats induced by PhIP possessed a characteristic mutation, a guanine deletion at 5'-GGGA-3' in Apc gene (52,53). This type of mutation is also found in the p53 gene of human cancers, and it is speculated that 3–10% of the p53 mutations detected in human cancers could be ascribable to PhIP (5). The gene mutations by the deletion of guanine at GGGA sequence may be explained by GGG sequence-specific DNA damage observed in this study.

Oral administration of PhIP to rats increased the 8-OH-dG level in mammary gland (14). PhIP-promoted rat mammary carcinogenesis was inhibited by feeding of isoflavone mixture including antioxidant genistein and daidzein (15). We consider that SOD may increase the frequency of mutations due to DNA damage induced by PhIP and thus increase its carcinogenic potential. In conclusion, oxidative DNA damage may play an important role in carcinogenicity of PhIP, in addition to the previously reported PhIP-DNA adduct formation.


    Notes
 
1 To whom correspondence should be addressed at: Department of Hygiene, Mie University School of Medicine, 2-174, Edobashi, Tsu, Mie, 514-8507, Japan Email: kawanisi{at}doc.medic.mie-u.ac.jp Back


    Acknowledgments
 
This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 20, 2001; revised January 25, 2002; accepted January 28, 2002.