Long Evans Cinnamon Rats DNA Damage during ...

Evidence of Alterations in Base Excision Repair of Oxidative DNA Damage during Spontaneous Hepatocarcinogenesis in Long Evans Cinnamon Rats Sujata Choudhury, Ronghe Zhang, Krystyna Frenkel, et al. Cancer Res 2003;63:7704-7707.

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[CANCER RESEARCH 63, 7704 –7707, November 15, 2003]

Evidence of Alterations in Base Excision Repair of Oxidative DNA Damage during Spontaneous Hepatocarcinogenesis in Long Evans Cinnamon Rats Sujata Choudhury,1 Ronghe Zhang,2 Krystyna Frenkel,2 Toshihiko Kawamori,3 Fung-Lung Chung,1 and Rabindra Roy1 1 2

DNA Repair Laboratory, Mechanism of Carcinogenesis Program, American Health Foundation Cancer Center, Institute For Cancer Prevention, Valhalla, New York; Department of Environmental Medicine, New York University School of Medicine, New York, New York; and 3National Cancer Center Research Institute, Tokyo, Japan

ABSTRACT The Long-Evans Cinnamon (LEC) rat, an animal model for Wilson’s disease, is an inbred mutant strain, which because of the genetic copper metabolism disorder develops hepatitis ⬃4 months after birth, followed by chronic hepatitis later in life, and eventually all of the surviving animals from liver injury and hepatitis develop spontaneous hepatocellular carcinomas. This animal model also shows that the generation of reactive oxygen species and the accumulation of oxidative damage in the liver DNA has significantly increased over the lifetime of LEC versus the wild-type Long-Evans Agouti (LEA) rats. Thus, the LEC rats having this genetically induced oxidative condition are proved to be very useful model for the study of endogenous DNA lesions and their relation to spontaneous carcinogenesis. In this study, we tested the hypothesis that differences do exist between these two rat strains in respect to their capacity to repair oxidative DNA base modification, which could explain the elevation of endogenous oxidative damage in the LEC rat liver DNA. We found that both the activity and expression at the protein and RNA levels of major DNA glycosylases, endonuclease III and 8-oxoguanine DNA-glycosylase, which initiate the excision and repair of oxidized bases, were significantly altered during the acute (16 –18 weeks) and early chronic (24 weeks) phases of hepatitis. Enzyme levels were restored in the later period of chronic hepatitis (week 40) in the LEC rat liver as compared with the age-matched LEA rats. This early reduction in the capacity to repair oxidative DNA base damage could have contributed to the accumulation of mutagenic adducts in liver DNA. These findings show for the first time in an animal model that acute hepatitis impairs the repair of oxidative DNA base damage and strongly suggest that the repair of endogenous DNA adducts plays a critical role in the development of spontaneous hepatocellular carcinoma in LEC rats.

INTRODUCTION Oxidative stress is arguably the most important cause of cellular genotoxicity, which is thought to cause diseases such as cancer and neurological disorders. Recent studies have linked oxidative stress and chronic inflammation with an increased risk of cancer (1). Certain diseases, characterized by oxyradical overload, such as WD4 also have been associated with the higher risk of liver cancer (2). The LEC rat, an animal model for WD, proved to be very useful in studying mechanisms of spontaneous carcinogenesis. LEC rats are a mutant Received 5/22/03; revised 9/18/03; accepted 10/2/03. Grant support: NIH RO1 Grants CA80917 (to R. R.), CA43159 (to F. L. C.), and CA37858 (to K. F.), National Institute of Environmental Health Sciences Center Grant ES 00260 (to K. F.), and National Cancer Institute Cancer Center Grant P30 CA 17613. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Present address: Toshihiko Kawamori, Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425. Requests for reprints: Rabindra Roy, DNA Repair Laboratory, Mechanism of Carcinogenesis Program, American Health Foundation Cancer Center, Institute For Cancer Prevention, 1 Dana Road, Valhalla, NY 10595, Phone: (914) 789-7130; Fax: (914) 5926317; E-mail: [email protected]. 4 The abbreviations used are: WD, Wilson’s disease; LEC, Long-Evans Cinnamon; LEA, Long-Evans Agouti; HCC, hepatocellular carcinoma; 8-oxoG, 8-oxoguanine; OGG, 8-oxoguanine DNA glycosylase; ROS, reactive oxygen species; BER, base excision repair; AP, apurinic/apyrimidinic; DHU, dihydrouracil; RT-PCR, reverse transcriptionPCR.

strain of Long-Evans rats and are genetically predisposed to spontaneous HCCs as compared with the wild-type control LEA rats. As with WD, the LEC rats accumulate excess copper in the liver and develop hepatitis ⬃4 months after birth, followed by chronic hepatitis at a later age, and eventually ⬃1 year later develop HCC (3). This model also showed that the generation of ROS, lipid peroxidation, and accumulation of oxidative damage (8-oxoG) and cyclic etheno adducts in DNA was significantly increased over the lifetime in the livers of LEC but not of LEA rats (4 – 6). DNA adduct levels peaked in 18-week-old rats during the acute hepatitis period, and the trend of that increase paralleled the levels of copper, ROS production, and lipid peroxidation measured in the liver. It is not yet known whether the differences in DNA repair capacity of these two rat strains contribute to this elevation of the oxidative and cyclic adducts in liver DNA of LEC rats. It has been recently shown that DNA strand breaks, as well as hepatocellular preneoplastic foci, also peaked during this period of hepatitis (7). Moreover, this study showed higher cell proliferation and relatively lower apoptosis during acute hepatitis stage. To ward off the deleterious effects of oxidized bases, organisms (including humans) have developed efficient repair mechanisms. In general, the oxidized base lesions are removed from DNA by the BER pathway that is initiated by DNA glycosylase/AP lyases, which not only catalyze the removal of the base lesions but also cause strand cleavage at the resulting AP sites via ␤-elimination by their associated AP lyase activity (8, 9). Among such oxidatively damaged bases, a series of structurally diverse toxic or mutagenic oxidized pyrimidines, including thymine glycol, 5-hydroxy cytosine, DHU and others, are repaired by endonuclease III (NTH1; Refs. 10 –12), whereas the OGG repairs the oxidized purine (8-oxoG; Ref. 13). Subsequent repair steps include removal of the resulting 3⬘-␣, -␤-unsaturated aldehyde by the phosphodiesterase activity of AP-endonuclease, filling the resulting DNA gap by a DNA polymerase, and finally, sealing the repaired strand by DNA ligase (9, 14). In the present study, we have investigated the excision repair capacity of oxidative damage, a potentially protective cellular mechanism. Lack of such a repair capability can cause accumulation of this type of DNA base damage. We found that both the activity and expression of major DNA glycosylases, NTH1 and OGG1, were simultaneously altered as a function of age in LEC rat liver extracts as compared with extracts from age-matched LEA rats. These findings show for the first time in an animal model that the repair mechanism of oxidative DNA damage is impaired by acute hepatitis, which is likely to contribute to the accumulation of the mutagenic adducts in liver DNA. These findings point to a critical role of the repair mechanism of endogenous oxidative DNA adducts in spontaneous HCC in LEC rats and, possibly, to human HCC. MATERIALS AND METHODS

Animals. The 4-week-old inbred male LEC rats were received as a gift from Dr. Kozo Matsumoto at the University of Tokushima (Tokushima, Japan). The LEA rats were bred and maintained at the animal laboratory of the National Cancer Center Research Institute (Tokyo, Japan). Rats were main7704


ALTERATION OF BER OF OXIDIZED BASES DURING HCC

tained under standard conditions with temperature and light control and were messages for NTH1, OGG1, and an internal marker gene ␤-actin were amplifed a standard diet of grain-based NIH-07 pellets. The animals were sacrificed fied using the PCR Master Mix kit (Promega). The PCR conditions and the at various ages and the liver tissues were preserved at ⫺80°C until use. The sequences of the primers for OGG1 and ␤-actin were followed according to the LEA rats were sacrificed at the National Cancer Center Research Institute; the published procedures (16), whereas the conditions for NTH1 were a single cycle of denaturation at 94°C for 5 min, then 36 cycles of denaturation at 94°C liver tissues were flash frozen and shipped to our laboratory in dry ice. Preparation of Liver Extracts. Liver extracts were prepared from LEA for 30 s, annealing at 64°C for 30 s and extension at 72°C for 30 s, followed and LEC rats after a modified published procedure (15). Briefly, liver tissues by a single cycle of final extension at 72°C for 10 min. Because the cDNA for (12 g) from LEC and LEA rats of various ages were minced into small pieces, rat NTH1 is not yet cloned, we designed the primers for RT-PCR of NTH1 in washed thoroughly with PBS, and homogenized. After centrifuging the homo- rat livers based on the highly conserved cDNA sequences of humans, mice, genate, the pellet was suspended in chilled buffer A [10 mM HEPES (pH 7.9), and Escherichia coli. Thus, the predicted size of the NTH1 RT-PCR product 10 mM KCl. 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and protease inhibitor was 330 bp. Using this primer set (sense, 5⬘-GCGCGGGGCCTGACGGTGmixture; Roche Biochemicals] and allowed to swell on ice for 15 min. The GAC-3⬘ corresponding to 85–105 nucleotides; and the antisense, 5⬘-GGCGswelled pellet was then mixed with 0.6% NP40 and 0.4 M NaCl and shaken in GCGCGGGTCTCCTCTG-3⬘ corresponding to 395– 414 nucleotides of human a rocking shaker for 15 min at 4°C. The lysate was then centrifuged, the NTH1), both human cells (HeLa, a human cervical cancer cell line) and supernatant (whole liver extract; 4 – 8 mg/ml) was stored at ⫺80°C in small LEA/LEC livers showed RT-PCR products of similar and expected sizes (Fig. aliquots and thawed only once for the in vitro activity assay to avoid inacti- 2B). However, to confirm the identity, we cloned the putative rat NTH1 vation attributable to repeated freeze-thaw cycles. These extracts used in the RT-PCR product in pCR2.1-TA cloning vector and sequenced. The sequence BER assays for oxidative DNA base damage are generally stable for even two matched 99.8% with human and 98% with mouse NTH1s. Other Methods. Proteins were quantified by the dye-binding method (Bioto three cycles of thawing and refreezing. Labeling of Oligonucleotides. Oligonucleotide Substrates: A DHU- Rad) using BSA as the standard. containing 56-mer oligonucleotide (DHU-56) with the sequence 5⬘-ATTATGCTGAGTGATATCCCTCTGGCCTTCGAACCCXACCTCAACCTC- RESULTS AND DISCUSSION TGCCCACC-3⬘ (where X represents DHU) was purchased from Operon. Oligonucleotides containing 8-oxoG with the sequence 5⬘-TCGAGGATCCTTo examine the capacity of the wild-type LEA versus the mutant GAGCTCGAGTCGACGXTCGCGAATTCTGCGGATCCAAGC-3⬘ (where LEC rats liver to repair DHU, an oxidized pyrimidine adduct, which X represents 8-oxoG) were obtained from Midland Certified Reagent Co. is acted upon by the BER pathway initiated by endonuclease III, a (Midland, TX). The complementary oligonucleotides containing A and C specific DNA glycosylase (10 –12), we used the liver extracts of opposite DHU and 8-oxoG were synthesized by the Recombinant DNA Labage-matched LEA and LEC rats of different ages. We measured the oratory Core Facility at the University of Texas Medical Branch at Galveston. of The oligonucleotides were purified on a sequencing gel. Both damage- NTH-mediated DNA glycosylase/AP-lyase activity by an incision th containing oligonucleotides were labeled at the 5⬘-end using T4 polynucleotide a 56-mer oligonucleotide containing a single DHU at the 27 position kinase and ␥[32P]ATP and were annealed to the respective complementary from the 5⬘-end. NTH1 excised the DHU base and cleaved 3⬘ to the oligonucleotides (damaged-strand:complementary ⫽ 1:1.7) to prepare 32P-end apurinic site in a concerted mechanism. The 26-mer incision product was then separated from the 56-mer substrate on a 10% sequencing labeled duplex oligonucleotides as described previously (10 –12). 8-OxoG and DHU Incision Assay. The 5⬘-labeled duplex oligonucleotide gel. The excision/incision activities at different ages of LEA and LEC substrates (DHU and 8-oxoG; 15 nM) were incubated at 32°C for 16 h with 50 rat liver extracts are presented in Fig. 1, A and B. We then tested the ␮g of protein of liver extracts derived from each of the LEA rats at various NTH1 expression by Western blotting (Fig. 2A) to ascertain whether ages (8, 14, 16, 18, 24, and 40 weeks) and LEC rats (8, 14, 16, 17, 18, 24, and the changes in activity correlate with those at the protein level. We 40 weeks) in a reaction buffer containing 50 mM HEPES-KOH (pH 7.9), 75 found that both the activity and level of NTH1 were simultaneously mM NaCl, 0.1 mg/ml BSA, 0.5 mM EDTA, and 1 mM DTT. The reactions were altered as a function of age in LEC rat liver extracts. Notably, in stopped by adding a solution containing 0.5% SDS and 20 ng/␮l tRNA at final concentrations. The proteins were extracted with phenol/chloroform and pre- contrast to the age-matched wild-type LEA rat liver extracts, both cipitated with ethanol. The precipitate was then dissolved in 10 ␮l of loading activity and amount of NTH1 decreased significantly (12–18-fold) dye (90% formamide, 0.03 N NaOH and 0.025% bromphenol blue, 0.025% during acute hepatitis (16 –18 weeks) and ⬃3-fold in early chronic xylene cyanol, and 4% glycerol). Control reactions were performed by incu- hepatitis (24 weeks) phases. These parameters were then restored in bating purified OGG1 and hNTH1 and DNA glycosylase/AP lyases known to the later period of the chronic hepatitis period (40 weeks) to the levels cleave 8-oxoG and DHU, with labeled oligonucleotides. Finally, all samples evident in the LEA rat extracts. Notably, the acute and chronic were heated at 95°C for 5 min, and 5 ␮l were loaded onto a denaturing 10% hepatitis phases described here for LEC rats are based on the previous polyacrylamide sequencing gel in 7 M urea and Tris-borate EDTA buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA). Radioactivity in the incised oligonucleotide was quantified by exposing the gel to a PhosphorImager (Molecular Dynamics). Western Blot Analysis. The liver extracts (50 ␮g) from the LEC and LEA rats of various ages were electrophoresed on 12% SDS-PAGE and transferred to a nitrocellulose membrane. As described earlier (12), Western blot analysis was carried out with the affinity-purified anti-hNTH1 polyclonal antibody (1:1000), anti-␤-actin (AC-15, 1:1000; Sigma) monoclonal antibody, and a horseradish peroxidase-linked antirabbit secondary antibody (1:2000; Amersham Pharmacia Biotech, Piscataway, NJ) by using the enhanced chemiluminescence technique (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer’s protocol. The protein bands were quantified by densitometric scanning of the X-ray film using the Epi Chemi II Darkroom (UVP, Inc., Upland, CA) and the attached labworks software (version 4.0) for Windows. Analysis of mRNA by RT-PCR. Total RNA was isolated from the livers Fig. 1. NTH1-mediated DNA glycosylase/AP-lyase activity in LEA (A) and LEC (B) of LEA and LEC rats using the RNA easy (Qiagen, Valencia, CA) kit rat liver extracts. The data presented are the mean ⫾ SD) values of three to five according to the manufacturer’s instruction. It was then used with RNase independent measurements from 2 rats of each strain. The details of the assay procedure are described under “Materials and Methods.” Substrate, 56-mer oligonucleotide containinhibitor (Promega, Madison, WI), reverse transcriptase (Superscript; Invitro- ing a single DHU; product, incised 26-mer oligonucleotide. C, quantitation of NTH1gen, Carlsbad, CA), and oligo(dT)18 for synthesis of a cDNA pool. The mediated excision/incision activity shown in A and B. 7705



Fig. 2. A, Western blotting of NTH1 protein expression in LEA and LEC rat liver extracts. B, analysis of NTH1 mRNA in LEA and LEC rat livers by RT-PCR. The details of the assay procedure are described under “Materials and Methods.” The NTH1/␤-actin ratio denotes the band intensities of NTH1 normalized over those of ␤-actin.

studies (3). The NTH1 protein amount and activity were also correlated with NTH1 mRNA, as shown by RT-PCR (Fig. 2B). Although the mRNA levels were reduced in both 14- and 17-week old LEC rats, interestingly, the NTH1 protein was still present in 14-week-old LEC rats (Fig. 2A). It is possible that the NTH1 mRNA is unstable, whereas the protein is stable in 14-week-old animals. It is also possible that mRNA expression is suppressed during an early stage of acute hepatitis, whereas the protein synthesized before this inhibition may have been still present throughout this period. We then examined the repair capacity in LEA and LEC liver extracts of an oxidized purine lesion, 8-oxoG, which is also repaired by the BER pathway initiated by OGG1 (13). We measured the OGG1-mediated DNA glycosylase/APlyase activity by incision of a 50-mer oligonucleotide containing a single 8-oxoG at the 27th position from the 5⬘-end. OGG1 excised the 8-oxoG moiety and cleaved 3⬘ to the apurinic site in a concerted action. The 26-mer incision product was then separated from the 50-mer substrate on a 10% sequencing gel; the excision/incision activities at different ages of LEA and LEC rat liver are presented in Figs. 3, A–C. Although, the basal activity of the OGG1 was 5– 6-fold less than that of the NTH1 in both the wild-type and mutant rat strains, the mRNA levels of the OGG1 at various ages of these rats were not much different from those of the NTH1 mRNA. This difference may be because of several reasons: (a) in addition to the NTH1, the DHU can also be excised by endonuclease VIII homologues (NEIL1 and 2; Refs. 17, 18); (b) after mRNA expression, OGG1 protein is expressed (the OGG1 protein level could not be tested in this study because of lack of availability of the appropriate antibody) at a similar level to NTH1, but to show the same incision activity as NTH1, OGG1 would require more assistance from coordinating enzymes such as APendonuclease (13), which could be rate limiting in LEA/LEC livers. However, similar to NTH1, the activity and mRNA expression level of OGG1 were also reduced during acute hepatitis in LEC rat liver but

not in LEA rat liver. Unlike NTH1, the activity of OGG1 was also reduced in LEC rat liver at the 14 weeks of age (Figs. 3, B–D). Thus, our findings show, for the first time, a modulation of repair of oxidative DNA bases in LEC rat liver. Yamamoto et al. (4) reported that the 8-oxoG levels were significantly increased in the liver DNA of 15-week LEC rats as compared with those detected in age-matched LEA rats. It appears that these adducts peaked during the onset of acute hepatitis, and the trend of their increase paralleled the copper accumulation and decrease in excision activity for oxidized purine and pyrimidines in LEC rat livers. Although the repair capacity was not measured, the generation of ROS and extensive elevation of oxidative DNA damage have elegantly been shown to be a primary cause of liver cancer in two different HCC animal models overexpressing either hepatitis B virus large envelope protein or c-myc/tumor growth factor ␣ (19, 20). OGG1-knockout mice have been shown to accumulate 8-oxoG in their genome and develop lung adenoma/carcinoma spontaneously 1.5 years after birth (21). Human OGG1 genes were found to be frequently mutated in lung adenocarcinoma (22); decreased repair activity of OGG1 attributable to alternative splicing of mRNA and genetic polymorphism have also been shown to be risk factors for lung adenocarcinomas (23, 24). On the other hand, NTH1-knockout mutation showed no detectable phenotypic abnormalities at relatively early ages (25) because at least three other DNA glycosylases of the NEIL family of proteins act as back-up DNA glycosylases for repairing similar endogenous adducts. However, the reduced BER of oxidative DNA damage caused by the inhibition of OGG and NTH enzymes have been shown to be primary etiologies for lung cancer during pulmonary adaptation to cadmium (26). Thus, these studies strongly support our findings that BER of endogenous DNA adducts play a critical role in carcinogenesis. It was recently shown that in LEC rat liver the preneoplastic foci were first evident at 24-week and peaked at 48-week age (7). Therefore, it is intriguing that the dramatic reduction of repair of oxidative base damage during 16 –18 weeks (Figs. 1–3) precedes the appearance of preneoplastic foci and signifies the contribution of repair of oxidized bases in tumorigenesis of LEC rat liver. Moreover, that study also showed higher cell proliferation and relatively lower apoptosis during the hepatitis period (7), supporting the notion that the reduction in the oxidative base damage repair (Figs. 1–3) is not merely because of cell killing. Taken together, both the reduction of DNA repair capacity, elevation of oxidative damage, and sustained cell proliferation during hepatitis may predispose individuals to increased mutation load and cancer risk (e.g., WD; Ref. 27). Moreover, the findings of restored NTH1 and OGG1 activities during the chronic hepatitis period raise the possibility that DNA repair enzymes may help the

Fig. 3. OGG1-mediated DNA glycosylase/APlyase activity in LEA (A) and LEC (B) rat liver extracts. The data presented are the mean (⫾SD) values of three to five independent measurements from 2 rats of each strain. Substrate, 50-mer oligonucleotide containing single 8-oxoG; product, incised 26-mer oligonucleotide. C, quantitation of OGG1-mediated excision/incision activity shown in A and B. D, analysis of OGG1 mRNA in LEA and LEC rat livers by RT-PCR. The details of the assay procedures are described under “Materials and Methods.” The OGG1/␤-actin ratio denotes the band intensities of OGG1 normalized over those of ␤-actin.

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cells to survive against additional deleterious oxidative stress. Therefore, these DNA repair enzymes in LEC rats appear to have possibly two contrasting roles: one in protecting normal cells against oxidative DNA damage, but once tumors are formed, in promoting the survival of tumor cells against additional oxidative DNA damage. To conclude, these findings indicate that the reduced repair capacity of oxidative damage by the BER pathway may play a critical role in the accumulation of mutagenic adducts in liver DNA of LEC rats and support a potential role of these adducts in spontaneous HCC of LEC rats. Moreover, these data signify that the deficiency of repair enzymes for oxidized purines (e.g., 8-oxoG) and pyrimidines (e.g., thymine glycol, 5-hydroxyuracil, and so on) may be a risk factor for developing hepatic cancer. Thus, these enzymes may be useful in the identification of oxidative carcinogens. They also may serve as early biomarkers in the identification of individuals at higher risk for hepatic cancer and, possibly, other inflammation-related cancers in humans. Finally, the accumulated copper in the liver may also have direct impact on the suppression of BER DNA glycosylases and thus the apparent epigenetic nature of the alterations in the NTH1 and OGG1 expression in LEC rat liver warrants the investigation of regulatory mechanisms of these important oxidative base damage repair genes. ACKNOWLEDGMENTS We thank Research Animal Facility at the American Health Foundation Cancer Center for their extensive help in the maintenance of LEC rats.

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