Immunohistochemical detection of a substituted 1,N2

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Linoleic acid,. 2-nonenal, succinic anhydride and o-phenylene diamine were obtained from ... Japan). 3,5-Diaminobenzoic acid was purchased from Aldrich (Milwaukee,. WI). ... (2 mM) and incubated in phosphate buffer (pH 7.4) at 37°C for 4 days. The ... The solution was then evaporated and the residue was dissolved in.
Carcinogenesis vol.23 no.3 pp.485–489, 2002

Immunohistochemical detection of a substituted 1,N2-ethenodeoxyguanosine adduct by ω -6 polyunsaturated fatty acid hydroperoxides in the liver of rats fed a choline-deficient, L-amino acid-defined diet Yoshichika Kawai, Yoji Kato1, Dai Nakae2, Osamu Kusuoka2, Yoichi Konishi2, Koji Uchida and Toshihiko Osawa3 Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan, 1School of Humanities for Environmental Policy and Technology, Himeji Institute of Technology, Himeji 670-0092, Japan and 2Department of Oncological Pathology, Cancer Center, Nara Medical University, Nara, 634-8521, Japan 3To

whom correspondence should be addressed Email: [email protected]

Endogenous lipid peroxidation products react with DNA and form exocyclic DNA adducts. The purpose of this study was to investigate the in vivo formation of 7-(2-oxo-heptyl)-substituted 1,N2-etheno-2⬘-deoxyguanosine adduct (Oxo-heptyl-εdG), one of the major products from the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with dG. The monoclonal antibody specific to Oxo-heptyl-εdG was prepared using a chemically synthesized conjugate of Oxo-heptyl-εdG and carrier protein as immunogen. The characterization showed that the obtained antibody (mAb6A3) is specific to the Oxo-heptyl-εdG moiety. Using the novel antibody, the presence of the Oxoheptyl-εdG adduct in vivo was immunohistochemically revealed in the liver of rats fed a choline-deficient, L-amino acid-defined diet, an endogenous carcinogenesis model, for 3 days. In addition, the Oxo-heptyl-εdG formation was confirmed in DNAs treated with peroxidized linoleic acid, arachidonic acid and γ-linolenic acid, respectively, suggesting that the hydroperoxides of ω-6 polyunsaturated fatty acids could be the potential sources of Oxo-heptylεdG formation in vivo. Collectively, the results in this study suggest the first evidence that the formation of Oxo-heptylεdG, the ω-6 lipid hydroperoxide-mediated DNA adduct, may be a potential biomarker for the early phase of carcinogenesis.

Introduction Much attention has been focused on the relationships between lipid peroxidation and carcinogenesis because lipid peroxidation products cause the covalent modification of nuclear bases and form mutagenic DNA adducts (1,2). During the lipid peroxidation process, lipid hydroperoxides are formed as the initial products, and the decomposition of the lipid hydroperoxides leads to the formation of aldehydes as the end products. Several aldehydes possess high reactivity against DNA bases, Abbreviations: ACR, acrolein; BSA, bovine serum albumin; CDAA, cholinedeficient, L-amino acid-defined; CSAA, choline-supplemented, L-amino aciddefined; dG, 2⬘-deoxyguanosine; ELISA, enzyme-linked immunosorbent assay; Gua, guanosine; HNE, 4-hydroxy-2-nonenal; 13-HPODE, 13-hydroperoxyoctadecadienac acid; KLH, keyhole limpet hemocyanin; MDA, malondialdehyde; ONE, 4-oxo-2-nonenal; PBS, phosphate-buffered saline; TBARS, thiobarbituric acid-reactive substances; TPBS, PBS containing 0.05% Tween-20. © Oxford University Press

in particular guanine, which is the most reactive base under physiological conditions. The 1,N2-substituted cyclic deoxyguanosine adducts by lipid-derived reactive aldehydes, such as malondialdehyde (MDA), acrolein (ACR) and 4-hydroxy-2nonenal (HNE), have been identified (3–5) and detected in rodent and human tissues (6–8). On the other hand, the lipid hydroperoxide-induced DNA adduct formation and site-specific cleavage of double-stranded DNA have been reported (9,10). However, the reaction mechanism of lipid hydroperoxides with DNA has scarcely been investigated so far. Based on this information, we started to investigate the reaction of lipid hydroperoxides with DNA components, and then identified one of the major products during the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with 2⬘-deoxyguanosine (dG) as the 7-(2-oxoheptyl)-substituted 1,N2-etheno-2⬘-deoxyguanosine adduct (Oxo-heptyl-εdG; Figure 1). This adduct has recently been reported from the reaction of 4-oxo-2-nonenal (ONE) with dG by Rindgen et al. (11). In view of the potential role of ethenotype DNA adducts for carcinogenesis (12,13), it may be important to investigate this novel etheno-adduct formation. However, the in vivo presence of this novel etheno adduct, Oxo-heptyl-εdG, still remains to be proven. In this study, we succeeded in the preparation of a monoclonal antibody specific to this etheno adduct. To examine the mechanism and pathophysiological significance of Oxo-heptylεdG formation in vivo, we applied the antibody to the immunohistochemical analysis of the liver of rats fed a cholinedeficient, L-amino acid-defined (CDAA) diet, an experimental model for endogenous carcinogenesis (14). Our results suggest that the formation of Oxo-heptyl-εdG in vivo may be a potential biomarker for oxidative DNA damage during the early stage of lipid peroxidation. Materials and methods Chemicals Calf thymus DNA, dG, acrolein, soybean lipoxygenase and bovine serum albumin (BSA) were obtained from Sigma (St Louis, MO). Keyhole limpet hemocyanin (KLH) was purchased from Pierce (Rockford, IL). Linoleic acid, 2-nonenal, succinic anhydride and o-phenylene diamine were obtained from Wako Pure Chemicals Industries, ICN Pharma (Aurora, OH). Horseradish peroxidase-labeled goat anti-mouse IgG was obtained from Cappel (Osaka, Japan). 3,5-Diaminobenzoic acid was purchased from Aldrich (Milwaukee, WI). The sodium salt of MDA was prepared by Dowex hydrolysis of malondialdehyde bis(diethyl acetal) as described previously (15). HNE was prepared by the acid treatment (1 mM HCl) of HNE dimethylacetal synthesized according to the procedure of De Montarby et al. (16). ONE was kindly provided by Dr Koichi Itakura (Aichi University of Education). 13-HPODE was enzymatically synthesized from linoleic acid by soybean lipoxygenase as described previously (17). Preparation of 13-HPODE- or aldehyde-modified DNA Calf thymus DNA (1 mg/ml) was mixed with 13-HPODE (2 mM) or aldehydes (2 mM) and incubated in phosphate buffer (pH 7.4) at 37°C for 4 days. The reaction was stopped by ethanol precipitation, and the precipitated DNA was washed with cold 70% ethanol. The amount of the DNA was determined by measuring the fluorescence intensity (ex 415 nm/em 505 nm) formed by the reaction of deoxyribose and 3,5-diaminobenzoic acid (18).

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Y.Kawai et al. ELISA The indirect non-competitive ELISA procedure has been described previously (17). Briefly, 100 µl of antigen in PBS were coated in wells and kept at 4°C overnight. After washing and blocking with 4% Block Ace (Dainihon Seiyaku, Osaka, Japan), 100 µl of the mAb6A3 (1/500 dilution) in PBS containing 0.05% Tween-20 (TPBS) was then added and the wells were incubated at 37°C for 2 h. After washing, 100 µl of anti-mouse IgG goat antibody peroxidase labeled (1/5000 in TPBS) was added and the wells were incubated at 37°C for 1 h. After washing, 100 µl of o-phenylene diamine solution (0.5 mg/ml containing 0.03% H2O2 in citrate–phosphate buffer) was added. The color developing reaction was stopped by the addition of 50 µl of 2 N H2SO4. The binding of the antibody to the antigen was evaluated by measuring the optical density at 492 nm. Competitive indirect ELISA was performed for estimating the crossreactivity of the low molecular weight compounds with the antibody. The Oxo-heptyl-εdG-BSA conjugate (0.05 µg/ well) was used as the coating antigen. For the competitive reaction, 50 µl of competitors in PBS were mixed with an equal volume of the antibody (1/1000 dilution) in PBS containing 4% BSA. The competitor solution was kept at 4°C overnight and 90 µl of the mixture was used as the primary antibody. The cross-reactivity of the antibody to the competitors was expressed as B/B0, where B is the amount of mAb6A3 bound to the coating antigen in the presence of the competitor and B0 in the absence of a competitor. Fig. 1. Structure of Oxo-heptyl-εdG adduct. Preparation of Oxo-heptyl-εdG, 7-(2-oxo-heptyl)-5,9-dihydro-9-oxo-3-β-Ddeoxyribofuranosylimidazo[1,2-a]purine Oxo-heptyl-εdG was prepared by reacting dG (2 mM) with 13-HPODE (2 mM) in phosphate buffer (pH 7.4). After 4 days, the reaction mixture was evaporated and freeze-dried, and then applied to a gel filtration column (Toyopearl HW-40, 2.8⫻50 cm; Tosoh, Tokyo) equilibrated with water. The fractions (10 ml/each) were monitored by absorbance at 230 nm using a Hitachi U-1100 spectrophotometer. The fractions containing Oxo-heptyl-εdG were evaporated and further purified by HPLC (Develosil ODS-HG-5, 8⫻250 mm, Nomura Chemical Co., Aichi, Japan). The elution was performed using 50% aqueous methanol at flow rate of 2 ml/min, monitoring the absorbance at 230 nm. The Oxo-heptyl-εdG was collected and purified by repeated HPLC. The obtained Oxo-heptyl-εdG was characterized by 1H-NMR and FAB-MS as reported previously by Rindgen et al. (11). Preparation of the monoclonal antibody to Oxo-heptyl-εdG To couple Oxo-heptyl-εdG to protein, the 5⬘-succinyl-Oxo-heptyl-εdG derivative (suc-Oxo-heptyl-εdG) was synthesized. Briefly, Oxo-heptyl-εdG (7.6 mg, 18.9 µmol) and succinic anhydride (3.8 mg, 37.8 µmol) were dissolved in pyridine (189 µl), and the mixture was kept at room temperature. After 2 days, the same amount of succinic anhydride was added and the mixture was kept overnight. The solution was then evaporated and the residue was dissolved in methanol. Suc-Oxo-heptyl-εdG was isolated by reverse-phase HPLC (Develosil ODS-HG-5, 8⫻250 mm) using 50% methanol containing 0.01% acetate at a flow rate of 2.0 ml/min monitoring at 230 nm. The obtained sucOxo-heptyl-εdG (2.3 mg, yield 24.2%) was identified by 1H-NMR and LC/MS measurements. The spectral data are as follows: 1H-NMR (CD3OD) (p.p.m.) 0.91 (t, J ⫽ 6.6 Hz, 3H, H-7⬙), 1.33 (m, 4H, H-5⬙, H-6⬙), 1.62 (m, 2H, H-4), 2.46 (m, 1H, H-2⬘a), 2.59 (m, 4H, CH2 of succinyl ester), 2.67 (m, 2H, H-3⬙), 2.86 (m, 1H, H-2⬘b), 4.15 (m, 1H, H-4⬘), 4.22 (s, 2H, H-1⬙), 4.30 (m, 1H, H-5⬘a), 4.36 (m, 1H, H-5⬘b), 4.62 (m, 1H, H-3⬘), 6.36 (m, 1H, H-1⬘), 7.04 (s, 1H, H-6), 8.05 (s, 1H, H-2); LC/MS (ESP⫹) m/z 504 (M ⫹ H)⫹. The carboxyl group of the obtained suc-Oxo-heptyl-εdG was conjugated to the ε-amino group of KLH or BSA by the carbodiimide procedure as described previously (19). The conjugate of suc-Oxo-heptyl-εdG and KLH (Oxo-heptylεdG-KLH) (0.6 mg/ml) was emulsified with an equal volume of complete Freund’s adjuvant. Six-week-old female Balb/c mice were intraperitoneally immunized with 100 µl of this emulsion. After 2 weeks, the mice were boosted with the Oxo-heptyl-εdG-KLH (0.2 mg/ml) emulsified with an equal volume of incomplete Freund’s adjuvant. In the final boost, 100 µl of the conjugate (0.5 mg/ml) in phosphate-buffered saline (PBS) was intravenously injected. Three days after the final boost, the mouse was killed and the spleen was removed for fusion with P3/U1 myeloma cells. The fusion was carried out by polyethylene glycol and the cells were cultured in hypoxanthine/ aminopterin/thymidine selection medium. Five days after the fusion, culture supernatants of the hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) using 13-HPODE-modified DNA and untreated DNA as the coating agents. The positive hybridomas were cloned by the limiting dilution method. After repeated screening and cloning, four specific clones were obtained. Among them, a clone (named mAb6A3, IgG2aκ) was used in the following experiment due to its specificity and high ability for cell growth.

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Immunohistochemical analysis Fischer 344 male rats, 6 weeks old, were obtained from Japan SLC, Hamamatsu, Japan. The CDAA diet and the control choline-supplemented, L-amino acid-defined (CSAA) diet were obtained from Dyets, Bethlehem, PA, USA. Details of the composition of the diets have been described previously (14). Five rats were fed the CDAA diet for 0 or 3 days and another group of rats fed the CSAA diet for 3 days was also used as the control. After the experimental period, the animals were killed and the livers were excised. The lipid peroxidation levels were evaluated by measurement of the thiobarbituric acid-reactive substances (TBARS) as described previously (20). For immunohistochemical staining, acetone-fixed, paraffin-embedded liver sections (4 µm thick) on silane-coated slide glasses were prepared. The sections were first incubated with 3% H2O2 for 10 min to prevent endogenous peroxidase activity and then with the primary antibody (1/500 dilution) for 2 h at room temperature. After washing, the sections were incubated with a secondary antibody (within LSAB2/HRP kit, Dako Japan, Kyoto.) for 10 min at room temperature. The sections were then washed again, and the avidin–biotin complex procedure with a LSAB2/HRP kit was conducted. After washing, the binding was detected with 3,3⬘-diaminobenzidine and then finally counterstaining was performed with hematoxylin. To confirm the specificity of immunostaining, competitive experiments were performed using the antibody pre-incubated with an excess of the Oxo-heptyl-εdG-moiety. Reaction of oxidized fatty acids with DNA Fatty acid (10 mM) was oxidized by 1 mM ascorbic acid and 0.01 mM FeSO4 in phosphate buffer at 37°C for 24 h. The oxidized fatty acid was mixed with an equal volume of DNA (1 mg/ml) and incubated at 37°C for 4 days. The modified DNA was collected by ethanol precipitation and used for ELISA. Statistics Statistical significance of the inter-group differences of means for multiple groups were determined using the Student Newman–Keuls multiple comparisons test after one-way ANOVA to determine variations among the group means followed by Bartlett’s test to determine the homogeneity of variance.

Results Preparation and the specificity of monoclonal antibody to novel etheno–dG adduct Incubation of 13-HPODE or ONE with dG gave Oxo-heptylεdG (Figure 1), a new type of substituted 1,N2-etheno–2⬘deoxyguanosine adduct, which was recently identified (11; our unpublished data). To verify the presence of the adduct in vivo, we tried to prepare a monoclonal antibody specific to Oxo-heptyl-εdG. To prepare the hapten–protein conjugate as the immunogen, the succinyl Oxo-heptyl-εdG derivative (sucOxo-heptyl-εdG) was chemically synthesized and the carboxyl group of suc-Oxo-heptyl-εdG was then coupled with the ε-amino group of KLH. Using the Oxo-heptyl-εdG-KLH conjugate, the monoclonal antibody to Oxo-heptyl-εdG was

Immunohistochemical detection of dG adduct by lipid hydroperoxides

Fig. 2. The characterization of the specificity of anti-Oxo-heptyl-εdG monoclonal antibody (mAb6A3). (A) The cross-reactivity of mAb6A3 (1/500 dilution) with various lipid-modified DNAs (1 µg/well) was estimated by non-competitive indirect ELISA. (B) The cross-reactivity of mAb6A3 with the Oxo-heptyl-εdG moiety was estimated by competitive indirect ELISA.

prepared as described in the Materials and methods. The specificity of the obtained monoclonal antibody (mAb6A3) was studied by ELISA. As shown in Figure 2A, mAb6A3 significantly reacted with the 13-HPODE- and ONE-modified DNA. Although this antibody did not show any specificity to the modified DNA by MDA, acrolein and 2-nonenal, respectively, the HNE-modified DNA was weakly recognized by the antibody. It has been suggested that ONE could be formed by oxidation of HNE during the incubation in phosphate buffer (21), therefore, it is also speculated that the antigenic Oxo-heptyl-εdG adduct can be generated during the reaction of HNE and DNA, although the detailed mechanism is still unknown. Competitive ELISA showed that mAb6A3 significantly recognized the Oxo-heptyl-εdG moiety (Figure 2B). The results suggest that the epitope of mAb6A3 is the Oxo-heptylεdG moiety. Immunohistochemical detection of the Oxo-heptyl-εdG adduct in vivo The presence of the Oxo-heptyl-εdG adduct in vivo has not yet been reported to the best of our knowledge. To examine the Oxo-heptyl-εdG formation in vivo, mAb6A3 was applied to the immunohistochemical staining in the liver of rats fed the CDAA diet. The CDAA diet is known as an experimental model for endogenous rat liver carcinogenesis associated with oxidative stress (14). It has been shown that 8-hydroxy-2⬘deoxyguanosine, an established pro-mutagenic oxidative DNA lesion, was significantly increased in livers by the CDAA diet and was involved in the development of putative pre-neoplastic lesions (14). The liver TBARS levels, a parameter of lipid peroxidation, were significantly increased by the feeding of the CDAA diet for 3 days as compared with the control groups (Figure 3A), showing that the CDAA diet caused significant lipid peroxidation in the liver within 3 days after the treatment. In agreement with the increasing lipid peroxidation, positive staining was observed in the liver of rats fed the CDAA diet for 3 days but not CDAA for 0 days (Figure 3B and C) and not CSAA for 3 days (data not shown). The staining was localized in nuclei, confirmed by counterstaining with hematoxylin. In addition, no positive staining was observed in the group fed the CDAA diet for 3 days with the mAb6A3 preabsorbed by the Oxo-heptyl-εdG moiety (Figure 3D), showing the specificity of the positive staining in the CDAA diet rats. Further evidence of the specificity was obtained using only the secondary antibody (without primary antibody) in which

Fig. 3. Immunohistochemical detection of Oxo-heptyl-εdG in the liver of rats fed the CDAA diet using mAb6A3. (A) The lipid peroxidation levels in the livers were evaluated by the measurement of the TBARS. Each point represents the mean ⫾ SD, n ⫽ 5. *P ⬍ 0.0005 (Welch), **P ⬍ 0.0001 (Student’s t-test). Sections of rat liver fed the CDAA diet for 3 days (B) and 0 days (C) were immunostained with mAb6A3 (1/500 dilution) as described in Materials and methods. (D) Competitive experiment using mAb6A3 preincubated with an excess of Oxo-heptyl-εdG. All sections were counterstained with hematoxylin.

no positive staining was observed (data not shown). These results show that the Oxo-heptyl-εdG moieties are generated in vivo and are correlated with the increasing lipid peroxidation levels. Oxo-heptyl-εdG formation by oxidized ω-6 polyunsaturated fatty acids In the previous section (Figure 3), the Oxo-heptyl-εdG formation was immunohistochemically shown in vivo, and associated with lipid peroxidation levels expressed as TBARS. It has been proposed that ONE, one of the major breakdown products of 13-HPODE, could form Oxo-heptyl-εdG during the reaction of 13-HPODE and dG (11,22). ONE is an analog of HNE, the representative end product derived from ω-6 polyunsaturated fatty acids. To investigate the potential sources of Oxo-heptylεdG formation in vivo, various oxidized polyunsaturated fatty acids were reacted with DNA, and the formation of Oxoheptyl-εdG was then evaluated by ELISA. As shown in Figure 4, the Oxo-heptyl-εdG formation was observed in the DNAs modified by oxidized ω-6 polyunsaturated fatty acids (linoleic acid, arachidonic acid and γ-linolenic acid) but not by oxidized ω-3 polyunsaturated fatty acids (α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). These results suggest that the hydroperoxides of ω-6 polyunsaturated fatty acids are potential sources of Oxo-heptyl-εdG formation in vivo. Discussion This report is the first demonstration of the in vivo formation of Oxo-heptyl-εdG. For the immunochemical detection of Oxo-heptyl-εdG, the monoclonal antibody specific to Oxoheptyl-εdG was prepared using the chemically synthesized hapten–protein conjugate as the immunogen. The characterization using ELISA showed that the obtained antibody (mAb6A3) specifically bound to the Oxo-heptyl-εdG moiety, but not to various aldehyde-modified DNA containing exocyclic DNA 487

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Fig. 4. Formation of Oxo-heptyl-εdG in the modified DNA by various oxidized fatty acids. Fatty acids (10 mM) were oxidized by the Fe2⫹/ascorbate system, and then reacted with DNA for 4 days at 37°C. Oxo-heptyl-εdG formation in the modified DNA was estimated by ELISA.

of ONE could contribute to DNA/RNA damage during the early phase of carcinogenesis. During the lipid peroxidation processes, lipid hydroperoxides are initially generated. Therefore, lipid hydroperoxide-mediated DNA adducts generate potentially in the early stage of lipid peroxidation. However, there has been little study that tried to prove this hypothesis. It has been shown that the CDAA diet causes subcellular injury due to oxidative stress in the early phase (14). The formation of Oxo-heptyl-εdG in CDAA diet fed rats was also observed after only day 1, in which the TBARS generation had not yet been observed (unpublished data), indicating that Oxo-heptyl-εdG formation may be one of the earliest DNA damage associated with lipid peroxidation. In conclusion, these results in this study raise the possibility that the DNA base modifications by lipid hydroperoxides may play an important role in the early phase of carcinogenesis, although the biological significance of Oxoheptyl-εdG formation are not yet fully demonstrated. The antibody mAb6A3 has become a good tool for the evaluation of the adduct in vivo. Acknowledgements

adducts (3–5). Using the specific antibody, we developed an immunohistochemical method to detect the Oxo-heptyl-εdG moiety in vivo. The intensive staining was observed in the liver section of rats fed a CDAA diet for 3 days. The positive staining was localized in the nuclei suggesting that the lipid peroxidation products generated by the treatment with the CDAA diet causes the nuclear base modifications in the early phase of carcinogenesis. The antibody mAb6A3 has also exhibited cross-reactivity with the reaction mixture of 13-HPODE with guanosine (Gua; RNA form) containing Oxo-heptyl-εGua adduct in vitro (unpublished data), suggesting the possibility that the Oxo-heptyl-εGua adduct in RNA could also be stained during the immunostaining (Figure 3). Yang et al. has immunohistochemically shown that the etheno-adducts, formed by 2,3-epoxy-4-hydroxynonanal (peroxide-mediated epoxide of HNE), are generated in DNA but not in RNA by treatment of the sections with DNase or RNase prior to the primary antibody treatment (13). Further investigations about Oxo-heptyl-εdG or Oxo-heptyl-εGua in the immunohistochemical analyses are also needed. Lipid hydroperoxides are known to be relatively short-lived. They are enzymatically and/or non-enzymatically metabolized to stable alcohols in vivo. They also react with metals to form reactive end products such as aldehydes. Therefore, the importance of lipid hydroperoxides to the covalent modifications of biological components has scarcely been investigated. Our demonstration in this study shows that the lipid hydroperoxide-derived intermediates could damage the nuclear DNA/ RNA and generate Oxo-heptyl-εdG/Gua in vivo. It has been proposed that the formation of Oxo-heptyl-εdG during the reaction of 13-HPODE with dG in vitro is mediated by ONE, because ONE is one of the major products of the metalcatalyzed breakdown of 13-HPODE (11, 22). We confirmed the ONE-derived formation of Oxo-heptyl-εdG in vitro, and also confirmed the cross-reactivity of mAb6A3 with the ONEmodified DNA (Figure 2A). Although the in vivo formation of ONE during the lipid peroxidation has not yet been proven, the immunohistochemical detection of Oxo-heptyl-εdG in this study suggests that the lipid hydroperoxide-derived production 488

We are very grateful to Dr Yasujiro Morimitsu and Yuji Ogino for their research support. We also thank Dr Koichi Itakura for providing ONE, and Kanako Ichihashi for technical support in preparing the monoclonal antibody.

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