Formation of 8-hydroxydeoxyguanosine and cell-cycle arrest in the rat liver via generation of oxidative stress by phenobarbital: association with expression profiles of p21WAF1/Cip1, cyclin D1 and Ogg1
Anna Kinoshita1,
Hideki Wanibuchi1,
Susumu Imaoka2,
Motome Ogawa1,
Chikayoshi Masuda1,
Keiichirou Morimura1,
Yoshihiko Funae2 and
Shoji Fukushima2,3
1 First Department of Pathology and
2 Department of Chemical Biology, Osaka City University Medical School, Abeno-ku, Asahi-machi 1-4-3, Osaka 545-8585, Japan
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Abstract
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To evaluate the risk of exposure to so-called non-genotoxic chemicals and elucidate mechanisms underlying their promoting activity on rat liver carcinogenesis the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), cytochrome P-450 (P-450) and hydroxyl radicals induction, DNA repair and alteration to cellular proliferation and apoptosis in the rat liver were investigated during 2 weeks of phenobarbital (PB) administration at a dose of 0.05%. Significant increase of hydroxyl radical levels by day 4 of PB exposure accompanied the accumulation of 8-OHdG in the nucleus and P-450 isoenzymes CYP2B1/2 and CYP3A2 in the cytoplasm of hepatocytes. Conspicuous elevation of 8-OHdG and apoptosis in the liver tissue were associated with reduction of the proliferating cell nuclear antigen (PCNA) index after 8 days of PB application. Thereafter, 8-OHdG levels decreased with an increase in mRNA expression for the 8-OHdG repair enzyme, DNA glycosylase 1 (Ogg1). Analysis with LightCycler quantitative 2-step RTPCR demonstrated induction of cyclin D1 (CD1) and p21WAF1/Cip1 mRNA expression on days 4 and 6, respectively, preceding marked elevation of PCNA and apoptotic indices. These results suggest that similar to genotoxic, non-genotoxic chemicals might induce reversible alteration to nuclear 8-OHdG in the rat liver after several days of continuous application; however, by a different mechanism. Increased 8-OHdG formation is caused by developing oxidative stress or apoptotic degradation of DNA and coordinated with enhanced expression of CD1 mRNA and cell proliferation, subsequent increase of p21WAF1/Cip1 mRNA expression, cell-cycle arrest and apoptosis, while activation of 8-OHdG repair mechanisms contributes to protection of tissue against reactive oxygen species-induced cell death.
Abbreviations: CYP2B1/2 and CYP3A2, 2B1/2 and 3A2 isoenzymes of cytochrome P-450, respectively; CD1, cyclin D1; CDK, cyclin-dependent kinase; DAB, 3,3'-diaminobenzidine; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; DMPO-OH, spin adduct of DMPO and hydroxyl radicals; ESR, electron spin resonance; GAPDH, glyceraldehyde-3-P dehydrogenase; HPLC, high-performance liquid chromatography; LC-RTPCR, LightCycler RTPCR; MnO, manganese oxide marker; NADPH, nicotinamide adenine dinucleotide phosphate reduced form; Ogg1, 8-oxoguanine DNA glycosylase 1; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; P-450, cytochrome P-450; PB, phenobarbital; ROS, reactive oxygen species; ssDNA, single-strand DNA.
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Introduction
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Non-genotoxic liver tumor promoter, phenobarbital (PB), a sedative and anti-convulsant used as an anti-epilepsy drug in humans, exerts promoting effects on hepatocarcinogenesis after suitable initiation in rats (1), and enhances the proliferation of carcinogen-exposed hepatocytes in vitro (2). Chronic administration of PB to rats and mice results in hepatocarcinogenicity (3,4). Depending on the time point, inhibition of cell proliferation, DNA synthesis and increased concentrations of TGF-ß1 have all been observed in the liver tissue (58). On the other hand, growth of pre-neoplastic lesions induced by PB might predominantly be attributable to an inhibition of programmed cell death/apoptosis or to induction of reactive oxygen species (ROS), oxidative DNA damage and genetic alterations by spontaneous errors in DNA replication and repair (911). Understanding the mechanism of PB-promoted tumor formation in rodents is an integral step toward more accurately assessing the potential risk of exposure to the non-genotoxic chemicals for human health.
ROS are involved in intracellular regulation of numerous factors, such as transcription factors kappa B, NF
B and AP-1, partly through protein kinase C (PKC) activation (1215). Oxygen radicals attack DNA bases and deoxyribose residues, producing damaged bases and single strand breaks or oxidize lipid and protein molecules, generating intermediates, which can react with DNA and form adducts. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is known as a marker of oxidative DNA damage, potentially involved in carcinogenesis in various experimental models (16). This modified base causes mutations, predominantly G to T transversions (17). The time- and dose-dependent generation of 8-OHdG in rat hepatic DNA have been demonstrated after single i.p. administration of genotoxic carcinogens including N-nitrosodiethylamine and aflatoxin B1 (16,18). In addition, 8-OHdG levels assessed after 1 week of dietary 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline application were also dose-dependently increased, although the effect was not observed at the end of the treatment period (19). These effects are suggested to be involved in initiation of hepatocarcinogenesis by genotoxic chemicals. On the contrary, it has been reported that only long-term high dose treatment with non-genotoxic liver tumor promoter PB results in the accumulation of 8-OHdG (20). As 8-OHdG in DNA is attributed to the production of oxygen radicals, especially hydroxyl radicals (21) and can be rapidly repaired (22), the actual level in the tissue is determined by changes in the rates of these processes. How PB treatment impacts on these remains unclear.
Recently, DNA damage has been shown to influence several genes involved in the cell-cycle checkpoint responses, inducing p53 (23), p21WAF1/Cip1 (24) and ATM (mutated in ataxia telangiectasia) (25). Although regulation of the cell cycle in response to DNA damage induced by genotoxic carcinogens has been extensively studied, there have been relatively few studies of this kind concerning the effects of non-genotoxic chemicals. Furthermore, links among oxidative stress and formation of DNA base modifications, cell-cycle regulation and DNA repair are not clearly understood.
To elucidate the risk of exposure to PB and investigate mechanisms underlying its promoting activity on rat liver carcinogenesis, in this study, our attention was focused on interrelations between the induction of oxidative stress, cytochrome P-450 (P-450) and hydroxyl radicals, formation of 8-OHdG and its repair and alterations to cellular proliferation and apoptosis in the livers of rats during continuous administration of the chemical. First, to analyze the formation of oxidative stress, the generation of hydroxyl radicals in the liver microsomal fraction was measured by electron spin resonance (ESR). Secondly, the cytochrome P-450 total content, activity and protein levels of isoenzymes CYP2B1/2 and CYP3A2 were examined. To evaluate the effect of oxidative stress on DNA damage and cell growth regulation, the levels of 8-OHdG, PCNA and apoptosis were determined by immunohistochemistry. In addition, the mRNA levels for the 8-OHdG repair enzyme, DNA glycosylase (Ogg1), were examined by semi-quantitative RTPCR. To investigate whether this non-genotoxic chemical exerts its carcinogenic effect in part by altering the cell-cycle regulation and cellular response to DNA damage, real-time LightCycler (LC) 2-step RTPCR was used for the quantitative determination of cyclin D1 (CD1) and p21WAF1/Cip1 mRNA expression.
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Materials and methods
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Chemicals
PB sodium salt (CAS no. 57-30-7) (purity
98%) was purchased from Wako Pure Chemicals Industries (Osaka, Japan) and other reagents from Wako Pure Chemicals Industries or Sigma (St Louis, MO). The spin-trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was obtained from Labotec Co. (Tokyo, Japan).
Animals
Five-week-old male Fisher 344 rats (Charles River, Japan, Hino, Shiga, Japan) were quarantined for 1 week before the start of the experiment. Animals were housed in an animal facility maintained on a 12 h (08:0020:00) light/dark cycle, at a constant temperature of 25 ± 1°C and relative humidity of 44 ± 5% and were given free access to tap water and food (Oriental MF powder diet, Oriental yeast Co., Tokyo, Japan).
Experimental design
Forty-eight male 6-week-old F344 rats were randomized into two groups, each including control and experimental subgroups. PB (0.05%) was administered in MF powder diet to rats for 1, 2, 4, 6, 8, 10, 12 and 14 days. At each time point three animals each in the experimental and control groups were killed. The numbers were selected in order to be able to complete the killing in 1 h. Animals were anesthetized with diethyl ether. The peritoneal cavity was then opened, and the portal vein was cannulated using an Insyte-WTM Vialon E I.V. catheter (Becton Dickinson Infusion Therapy Systems, Sandy, UT). The vena cave inferior was then cut and perfusion was begun in situ at 2 ml/min with ice-cold 1.15% KCl buffer (1.15% KCl, 1 mM EDTA, 0.25 mM PMSF) at room temperature. This was continued for 8 min, following which the liver was immediately removed. Separate portions were fixed in Bouin's solution and 10% buffered formalin. In addition, samples were frozen in liquid nitrogen and stored at 80°C for molecular analysis. The remaining liver tissue was immediately processed for microsome isolation, as described previously (26).
P-450 examination
The rat liver microsomal fraction was used for the examination of P-450 content (27), and activity determination in terms of P-450-mediated hydroxylation of testosterone, by high-performance liquid chromatography (HPLC) (28) and western blotting (29).
ESR
DMPO was employed as a spin-trapping agent with ESR signals measured using a quartz flat cell (inner size, 60x10x0.31 mm) and a JES-TE200 ESR Spectrometer (Japan Electronics Datum Co., Osaka, Japan). The reaction was started by addition of 10 mM nicotinamide adenine dinucleotide phosphate reduced form (NADPH) to liver microsomes (100 µg) in 0.1 M potassium phosphate buffer (pH 7.4) with 100 mM of the superoxide dismutase inhibitor diethyldithiocarbamate, followed by incubation at 37°C for 5 min. ESR spectra were recorded 30 s after addition of DMPO to the DMPO-free pre-incubation mixture with a reactive sample volume of 300 µl. As negative controls, several samples were run without addition of NADPH to the reaction mixture. After recording, the signal intensity of the spin adduct of DMPO and OH· (DMPOOH) was evaluated from the peak height of the third signal of the quartet and normalized relative to the standard signal intensity of the manganese oxide marker (MnO).
Localization of 8-OHdG
Liver sections fixed in Bouin's solution were used for immunohistochemical assessment of 8-OHdG. All sections were prepared 1 day before the immunohistochemical examination, which was performed in quintuplicate. After deparaffinization, sections were sequentially pre-treated with 0.3% H2O2 in distilled water for 30 min, 0.05 M NaOH in 40% ethanol for 12 min and 250 µg/ml RNAse A for 60 min at 37°C. Then they were exposed to avidin, then biotin, for 20 min each step to block non-specific binding of Avidin/Biotin System reagents (Vector Blocking Kit, cat. SP-2001, Vector Lab., Burlingame, CA). The sections were subsequently incubated with 10% horse blocking serum for 20 min at 37°C to prevent background staining before incubation with diluted anti-8-OHdG mouse monoclonal antibody (IgG1, 20 µg/ml) 1:500 overnight. Immunoreactivity was detected by the ABC Method (Vectastain Elite ABC kit, Vector Lab.) with color development using 3,3'-diaminobenzidine (DAB). Mayer's hematoxylin was then added as a counterstain for 1 min. Negative controls were immunostained as above, but with primary serum instead of the anti-8-OHdG antibody. To check the possibility of the immunohistochemical detection of cells that might be susceptible to oxidation by H2O2, liver sections were pre-treated with different concentrations of H2O2 and staining intensity has been compared. Furthermore, control detection of liver DNA 8-OHdG levels has been performed by HPLC method (30). Total hepatocyte numbers in areas of liver sections and all positive nuclei were counted with the aid of a color image analyzer (IPAP, Sumika Technos, Osaka, Japan) and results were expressed as the number of positive nuclei per 1000 cells.
Double immunohistochemistry for 8-OHdG and P-450
Double staining for 8-OHdG and P-450 isoenzymes was performed using anti-8-OHdG mouse monoclonal antibody (1:500) and polyclonal rabbit anti-CYP2B1/2 (1:1500) and anti-CYP3A2 (1:1000) (IgG, 100 µg/ml) antibodies. After deparaffinization sections were sequentially pre-treated with 0.3% H2O2 in distilled water and goat serum to block the background staining, they were exposed to polyclonal rabbit anti-CYP antibodies overnight (4°C). The sites of peroxidase binding were demonstrated with alkaline phosphatase [Vectastain ABC-AP kit, Vector Red (SK-5100)] solution. Thereafter, sections were sequentially treated with 0.2 M glycine, pH 2.2, for 2 h to remove immune complexes. Immunohistochemistry for 8-OHdG was performed as described above, except with the 0.3% H2O2 treatment. The red color cytoplasm of hepatocytes reflected binding of rabbit polyclonal primary antibody to cytochrome P-450, while brown or black staining of nuclei showed a positive immunoreaction of monoclonal primary antibody with 8-OHdG.
Detection of apoptosis
Formation of single-stranded DNA (ssDNA) in the liver of F344 rats treated with 0.05% PB, which occurs during programmed cell death/apoptosis, was detected in Bouin-fixed sections with polyclonal rabbit anti-ssDNA antibody (IgG, 100 µg/ml, Dako Japan Co., Kyoto, Japan) (31). After deparaffinization, sections were sequentially pre-treated with 0.3% H2O2, washed with Tris buffer solution (TBS) and incubated with horse blocking serum to prevent background staining. Exposure to anti-ssDNA antibody was at 1:400 dilution for 60 min at room temperature. Immunoreactivity for the primary antibody was detected with goat anti-rabbit immunoglobulins conjugated to peroxidase labeled-dextran polymer in TrisHCl buffer containing a carrier protein and an anti-microbial agent (Dako EnVision+TM Peroxidase, Rabbit kit, Dako). Color was developed using DAB. The apoptotic index was estimated as for 8-OHdG, as the number of positive nuclei per 1000 cells.
Double immunohistochemistry for 8-OHdG and apoptosis
Double staining for 8-OHdG and apoptosis has been performed as described above using alkaline phosphatase (Vectastain ABC-AP kit, Vector Blue) solution for the immunohistochemical detection of apoptosis, and ABC method with color development by DAB for the determination of 8-OHdG. Brown- and blue-stained nuclei reflected binding of primary antibody against 8-OHdG and ssDNA, respectively, while deep black staining showed positive immunoreaction with both 8-OHdG and ssDNA.
PCNA
Formalin-fixed sections were incubated with monoclonal anti-PCNA antibody (PC-10, IgG2a; Dako) at 1:500 dilution, followed by ABC-peroxidase procedures. The substrate was hydrogen peroxide with the coloring agent DAB and the PCNA index was assessed as for 8-OHdG and apoptotic indices.
RNA preparation
Total RNA was isolated from rat liver (pieces <5 mm in diameter) using Isogen (Nippon Gene, Toyama, Japan) (32). RNA was isopropanol precipitated, dissolved in DEPC-treated distilled water and kept at 80°C until use. RNA concentrations were determined with a spectrophotometer (Ultraspec 3000, UV/Visible Spectrophotometer; Pharmacia Biotech, Tokyo, Japan). Reverse transcription of 3 µg of total RNA was performed with Oligo-dT primer, and cDNA samples were stored at 20°C until assayed.
Analysis of Ogg1 mRNA induction
The samples prepared for PCR analyses were amplified in a 35 cycle-PCR reaction with DNA glycosylase (Ogg1) mRNA-specific primers as described by Tsurudome et al. (33). To perform RTPCR semi-quantitatively, we confirmed that PCR products were increased linearly with the input of cDNA. Glyceraldehyde-3-P dehydrogenase (GAPDH) mRNA expression was used as an internal standard. PCR products were separated on 3% NuSieve agarose gels, and analyzed using a FMBIO II Multi-View Image Analyzer Scanning Unit (Hitachi, Japan).
Standards for RTPCR
Using the RNA extracted from the livers of rats without any chemical treatment as a template, CD1, p21WAF1/Cip1 and GAPDH were amplified by RTPCR and subcloned in pT-AdV vector plasmid using an AdvanTAgeTM PCR cloning Kit (Clontech Laboratories, CA, USA). Finally, amplified plasmids were isolated with QIAGEN Plasmid Mini Kit (Funakoski Co., Tokyo, Japan), measured in a spectrophotometer, and molecule concentrations were calculated. Sequence analysis was performed to check the obtained plasmid quality. Serial dilutions ranging from 109 to 102 molecules were then prepared.
Real Time quantitative LightCycler (LC) PCR
PCR for CD1, p21WAF1/Cip1 and internal control, GAPDH, was performed using single pairs of primers and fluorescent probes (Table I
) and a LightCyclerFastStart DNA Master HybProbes Kit (Roche Molecular Biochemicals, Germany). Probes were designed to hybridize to the antisense strands of transcripts and were labeled with 6-carboxy-fluorescein phosphoramidite at the 5' end, and, as a quencher, 5-carboxy-tetramethyl-rhodamine (Nihon Gene Research Lab., Sendai, , Japan). The 20 µl PCR reaction mix contained 2 µl 10 PCR buffer, 8 mM (CD1) or 2.5 mM (p21WAF1/Cip1) MgCl2, 0.2 mM dNTP, 0.5 µM of each primer and 0.2 µM probe, 2 U of FastStart Taq DNA polymerase and 100 ng of sample cDNA. PCR amplification began with a 10 min pre-incubation step at 95°C, followed by 45 cycles of denaturation at 95° C for 0 s, annealing at 58°C (CD1) or 59°C (p21WAF1/Cip1) for 15 s, and elongation at 72°C for 17 s (CD1) or 11 s (p21WAF1/Cip1). The relative concentration of PCR product derived from the target gene was calculated using software of the LightCycler System. A standard curve for each run was constructed by plotting the crossover point against the log (number of starting molecules). The number of target molecules in each sample was then calculated automatically by reference to this curve. Results were expressed relative to the number of GAPDH transcripts used as an internal control. Some amplification products performed in the LightCycler were checked by electrophoresis on 3% ethidium bromide-stained agarose gels. All experiments were performed in quintuplicate.
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Table I. Nucleotide sequence for PCR primers, hybridization probes and conditions for amplification and sequence-specific detection of cDNA with the LightCycler System by real-time quantitative 2-step RT-PCR
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Statistical analysis
Statistical analysis of our data was performed using the Student's t-test according to Welch. All analyses were performed using the StatView-J 4.5 program (Berkeley, CA).
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Results
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Production of oxidative stress
To assess oxidative changes induced by PB administration in rat liver we measured hydroxyl radical levels and total content of cytochrome P-450 along with protein and activity of its two isoenzymes, CYP2B1/2 and CYP3A2, in the microsomal fraction. Formation of hydroxyl radicals was detected by the ESR technique. Figure 1A
shows ESR spectra for DMPOOH spin adducts in liver microsomal fraction at day 8 after beginning administration of 0.05% PB. The spectrum is in agreement with that reported earlier for spectrum of DMPOOH adduct (34). The signal intensity of the third OH· peak in the quartet was normalized relative to the standard signal intensity of the MnO peak used as an internal control. As shown in the Figure 1B
, DMPOOH adduct was significantly enhanced by day 4 of PB exposure, reached a maximum by day 8 (82.06 ± 5.99%; P < 0.001), and thereafter remained stationary. Only at day 8, very low levels of DMPOOH adduct were detected in liver microsomes of PB-treated rats without addition of NADPH to the reaction mixture (Figure 1A
).

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Fig. 1. Generation of hydroxyl radicals determined by ESR. DMPO was used as a spin-trapping agent. (A) DMPOOH spectra with liver microsomal fraction from rats treated with 0.05% PB for 8 days. Very low levels of DMPOOH adduct were detected in liver microsomes of PB-treated rats without addition of NADPH to the reaction mixture. (B) DMPOOH levels after application of PB. The signal intensity of the third OH· peak in the quartet was normalized relative to the standard signal intensity of MnO peak used as an internal control. Values are mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.001 (Student's t-test).
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Spectrophotometric and western blot analyses of liver microsomes revealed a time-dependent significant elevation of total P-450 content and protein levels of two P-450 isoenzymes inducible by PB, CYP2B1/2 and CYP3A2 (data not shown). Similar to hydroxyl radical levels, CYP2B1/2 and CYP3A2 activity was rapidly increased until day 8, and thereafter remained constant with insignificant deviation (Figure 2
). The activity of CYP3A2 appeared to be higher than that of CYP2B1/2 in contrast to the protein levels of these P-450 isoforms.

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Fig. 2. Induction of cytochrome P-450 isoenzymes CYP2B1/2 and CYP3A2 by administration of 0.05% PB. Activity levels were evaluated in terms of P-450-mediated hydroxylation of testosterone. The results are mean ± SD values (n = 3).
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Concomitant elevation of 8-OHdG and P-450
Results of immunohistochemical examinations are summarized in Table II
. Significant increase in 8-OHdG-specific immunoreactivity was detected on days 4, 6, 8, 10 and 12 post-treatment with 0.05% PB (Table II
). Positively stained hepatocytes were mainly localized around the central vein (Figure 3A and C
). Unexpectedly, on day 8, the level of 8-OHdG was enhanced up to the value of 18.79 ± 3.78 number/1000 cells, and 2 days later dramatically reduced, returning to the control level (0.14 ± 0.03 number/1000 cells) by day 14 (Table II
and Figure 4
). Furthermore, alterations of hydroxyl radicals and 8-OHdG levels were not strictly in parallel. Markedly increased until day 8, DMPOOH adduct levels thereafter remained unchanged, thus demonstrating a striking difference from the observed alteration of 8-OHdG (Figure 1B
and Table II
). To check the reproducibility of our immunohistochemical findings a further experiment was performed. Rats were treated with 0.05% PB for 6, 8 and 10 days and liver sections were immunohistochemically analyzed. Accumulation of 8-OHdG in the nuclei of hepatocytes at day 8 post-treatment and its reduction 2 days thereafter exactly reproduced the experimental results described above. Furthermore, elevation of 8-OHdG on days 4 (1.27 ± 0.19 8-OHdG/106 dG; P < 0.05), 6 (1.41 ± 0.14 8-OHdG/106 dG; P < 0.01), 8 (2.8 ± 0.05 8-OHdG/106 dG; P < 0.0001), 10 (1.25 ± 0.19 8-OHdG/106 dG; P < 0.05) and 12 (0.96 ± 0.16 8-OHdG/106 dG; P < 0.05) with the peak on day 8 was detected using the HPLC method and a good correlation with the results of immunohistochemistry was found. This was done in order to confirm that the original 8-OHdG levels in hepatocytes were detected by immunoassay and no damage was induced by H2O2 treatment used in the protocol of the immunohistochemical examination. Moreover, to check the possibility of the immunohistochemical detection of cells that might be susceptible to oxidation by H2O2 liver sections were pre-treated with different concentrations of H2O2, however, no difference in staining intensity was observed.
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Table II. Immunohistochemical assessment of 8-OHdG, apoptosis (ssDNA) and PCNA in liver sections of 0.05% PB-treated and normal rats
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Fig. 3. Double immunohistochemistry for 8-OHdG and P-450, and immunohistochemical assessment of apoptosis (ssDNA) in the livers of F344 rats treated for 8 days with 0.05% PB. (A) Immunohistochemistry for 8-OHdG and CYP 2B1/2 in a PB-treated (x100) and (B) control animal (x100); (C) 8-OHdG and CYP3A2 in PB-treated (x400) and a (D) control animal (x100). The red color cytoplasm of hepatocytes reflects binding of rabbit polyclonal primary antibody against cytochrome P-450, while brown or black stained nuclei showed positive immunoreactions for monoclonal primary antibody against 8-OHdG. Note: the increase of 8-OHdG in the nuclei of pericentrally localized hepatocytes most strongly stained for CYP2B1/2 and CYP3A2. (E) Immunohistochemical assessment of apoptosis (ssDNA) in PB-treated (x100) and (F) normal F344 rats (x100). As with 8-OHdG, positive immunoreactivity for ssDNA was found in the nuclei of pericentrally localized hepatocytes.
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Fig. 4. Co-ordinated changes of 8-OHdG, apoptosis (ssDNA) and PCNA in the livers of rats treated with 0.05% PB.
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Double staining for 8-OHdG and P-450 revealed an increase of CYP2B1/2 and CYP3A2 proteins in pericentrally localized hepatocytes, accompanied by 8-OHdG elevation in the nuclei (Figure 3AD
). Immunohistochemistry clearly demonstrated that an increase of 8-OHdG occurs primarily in hepatocytes strongly staining for CYP2B1/2 (Figure 3A and B
) and CYP3A2 (Figure 3C and D
) suggesting that the formation of oxidative base modifications is functionally dependent on the protein and activity levels of these P-450 isozymes.
Evaluation of apoptosis (ssDNA)
As shown in Table II
, 2.5- and 10-fold increase in the number of apoptotic cells was found at days 6 and 8, respectively, following PB administration. Only the nuclei of hepatocytes localized in the pericentral region were positively stained for ssDNA (Figure 3E and F
). Correlations were clearly apparent with alterations in 8-OHdG (Table II
and Figure 4
). Furthermore, the double staining for 8-OHdG and apoptosis revealed, that nuclei positively stained for apoptosis were always positive for 8-OHdG. However, only 70% of 8-OHdG-stained cells showed positive immunoreaction with antibody against ssDNA (Figure 5
). Apoptotic degradation of DNA was well associated with DNA oxidation. Similar to the case with nuclear 8-OHdG levels, enhancement of ssDNA formation at day 8 post-PB exposure was followed by sudden reduction. On day 10, the average number of apoptotic cells had decreased to a value significantly lower than that in the control group (Table II
). However, at days 12 and 14 no significant differences in the apoptotic index between control and experimental groups were detected.

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Fig. 5. Double immunohistochemistry for 8-OHdG and apoptosis (ssDNA) (x400). Brown and blue-stained nuclei reflected binding of primary antibody against 8-OHdG and ssDNA, respectively, while deep black staining showed positive immunoreaction with both 8-OHdG and ssDNA. Long arrownuclei positive for 8-OHdG and apoptosis, Arrow head8-OHdG positive cells.
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PCNA
A 2-fold elevation of the PCNA index reflecting changes in cell proliferation was found at day 6 after starting PB treatment (Table II
). Unexpectedly, it declined significantly at day 8. However, from day 10 of PB exposure, PCNA values slowly increased again.
Induction of Ogg1 mRNA expression
In order to elucidate the reason for the sudden reduction of nuclear 8-OHdG levels observed in our study, we investigated mRNA expression for the 8-OHdG repair enzyme, Ogg1. RTPCR semi-quantitative analysis revealed the time-dependent induction of Ogg1 mRNA (Figure 6
). Only small amounts of Ogg1 mRNA were detected in the non-treated controls. Significant increase of Ogg1 mRNA was observed beginning from day 6 of PB exposure, and the highest levels of expression were found at days 12 and 14.

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Fig. 6. Ogg1 mRNA levels in rat liver after administration of 0.05% PB. Significant increase of Ogg1 mRNA was observed beginning from day 6 of PB exposure, and the highest levels of expression were detected at days 12 and 14.
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Cell-cycle arrest
To determine whether PB exerts its carcinogenic effect in part by altering cell-cycle regulation, we investigated an expression of CD1 and p21WAF1/Cip1 mRNA by LC-RTPCR. Results were expressed relative to the number of GAPDH transcripts used as an internal control. The highest induction of CD1 mRNA expression at day 4 (0.45 ± 0.01; P < 0.0005) preceded the highest elevation of PCNA indices on day 6 (Figure 7A
and Table II
). This enhancement of CD1 mRNA expression was followed by lowest expression at day 6, prior to suppression of the PCNA index observed in our experiment at day 8 (Figure 7A
and Table II
). At days 8 and 10 the average copy numbers of CD1 transcripts again increased, reaching the control level, and thereafter again showed a tendency for enhancement. p21WAF1/Cip1 mRNA expression was significantly induced only at day 6 of PB exposure (0.42 ± 0.07; P < 0.005) at the time of increase in the PCNA index and prior to the elevation of 8-OHdG and apoptosis determined by immunohistochemistry (Figure 7B
and Table II
). These results are in good correlation with those observed by double staining for 8-OHdG and apoptosis, being an indication that p21WAF1/Cip1 mRNA expression might be increased in a certain percentage of hepatocytes, which are positively stained for both, 8-OHdG and apoptosis on day 8. Alterations of both CD1 and p21WAF1/Cip1 mRNA indicated that activity of ROS induced by PB treatment at a carcinogenic dose results in enhancement of cellular proliferation followed by cell-cycle arrest and apoptosis regulated by CD1 and p21WAF1/Cip1, which occur simultaneously with the increase of 8-OHdG.

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Fig. 7. (A and B) CD1 and p21 WAF1/Cip1 mRNA expression, respectively, in the livers of rats treated with 0.05% PB detected by real-time 2-step quantitative LightCycler PCR. GAPDH was used as an internal control. The number of target molecules in each sample was calculated automatically with reference to the standard curve (r = 0.99). Results were expressed initially as relative to the number of GAPDH transcripts used as an internal control. The data are mean ± SD values. *P < 0.01, **P < 0.005, ***P < 0.0005.
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Discussion
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The present study demonstrated a range of changes in cell proliferation, DNA damage and apoptosis-related parameters dependent on the period of PB exposure. While no formation of 8-OHdG in the rat liver was earlier found after up to 22 weeks of PB administration at a high dose (35), marked enhancement 8 days after starting the application was evident here. Previously, Murkofsky et al. (36) reported that treatment with 0.05% PB for 5 days in F344 rats induced cell proliferation, however, it did not affect steady-state levels of 8-OHdG in liver DNA. Our results indicated a conspicuous but short time 8-OHdG increase of nuclear localization in hepatocytes, which was associated with the rise in the intracellular hydroxyl radical level and in the beginning, with the elevation of cellular proliferating activity. In the present study, PB treatment was continued for 2 weeks with killing performed every 2 days, while the duration of previously published short-term experiments was no longer than 1 week. That gave us an opportunity to reveal the correlative changes in oxidative stress, proliferation, apoptosis, DNA damage and its repair, which occur in the rat liver during continuous PB exposure. Induction of Ogg1 mRNA expression suggested that one of the reasons for subsequent 8-OHdG decrease might be activation of the 8-OHdG repair. This has been shown to be activated in response to DNA damage generated by hydroxyl radicals and is attributable to glycosylase, endonuclease and lyase activity (22,37,38). Activation of 8-OHdG repair processes might explain the failure to detect 8-OHdG formation in the rat liver up to 22 weeks of PB application. Furthermore, changes of oxidative base modifications level observed in the present experiment are consistent with results demonstrating reversible alteration to liver DNA 8-OHdG after administration of genotoxic carcinogens, which is suggested to be potentially involved in initiation of hepatocarcinogenesis in rats (16,18,19). On the other hand, an induction of a significant and steady elevation of 8-OHdG is thought to be essential for the activation of carcinogenic properties of the cells (21). Recently, the longer maintenance of high levels of 8-OHdG in liver DNA is explained by the exhaustion and/or disturbance of the DNA repair system by the administration of carcinogens (16). It is thus conceivable that early increment of 8-OHdG in the nuclei of hepatocytes, induced by non-genotoxic chemicals via generation of oxidative stress, might influence the carcinogenic potential of initiated cells with already disrupted DNA repair producing stronger damage to DNA and promoting hepatocarcinogenesis.
Coordinated accumulation of 8-OHdG in the nucleus and CYP2B1/2 and CYP3A2 in the cytoplasm implied that increase of DNA damage after treatment with 0.05% PB in rat is caused by production of oxidative stress, related to increase in protein and activity levels of these two P-450 isoenzymes. Our results obtained by biochemical examination corresponded with the data reported previously on the induction of CYP2B1/2 and CYP3A2 by PB (10).
Interestingly, immunohistochemical examination of rat livers after 8 days of PB treatment showed the high levels of concordance between induction of 8-OHdG and apoptosis (ssDNA), which were inversely correlated with the PCNA labeling index (Figure 4
). Furthermore, double immunohistochemistry for 8-OHdG and apoptosis demonstrated, that in ~70% of cells with damaged DNA nuclei were apoptotic, suggesting that 8-OHdG elevation induced DNA fragmentation. However, the observation that oxidative DNA damage is mostly observed in the apoptotic cells could also mean that the oxidative degradation of DNA is the consequence rather than the cause of apoptosis. To investigate the mechanisms we analyzed mRNA expression of CD1 and p21WAF1/Cip1, genes involved in cell-cycle regulation and cellular response to DNA damage.
CD1 plays a key regulatory role during G1 phase of the cell cycle and its gene is amplified and overexpressed in many cancers. Recent studies have indicated that the induction of both CD1 and p21WAF1/Cip1 may contribute to wild type p53-mediated G1 growth arrest (39). p21WAF1/Cip1, a cyclin-dependent kinase (CDK) inhibitor and downstream effector of p53-mediated growth suppression, which is capable of linking the DNA damage response pathway with the cell-cycle machinery, contributing to DNA repair and maintaining genomic integrity (40,41). Recent studies have shown that it is also regulated through a p53-independent pathway (42,43). A unique feature of p21WAF1/Cip1, which distinguishes it from other CDK inhibitors, is its ability to associate with PCNA and induce cell-cycle arrest in G1 and G2 phases (44,45). It has been further shown to function as a tumor suppressor through its ability to control cell-cycle progression (46).
In this investigation, analysis with LC-RTPCR revealed the increased mRNA expression of CD1 and p21WAF1/Cip1 by 4 and 6 days, respectively, after starting 0.05% PB administration. p21WAF1/Cip1 mRNA alteration was observed during significant increase of PCNA, and prior to the elevation of 8-OHdG and apoptosis. These results suggested that application of 0.05% PB to non-initiated rats for 1 week results in an induction of CD1 mRNA and cellular proliferation, which are followed by p21WAF1/Cip1-dependent G1 and G2 arrest. The last phenomenon is likely to be related to the p21WAF1/Cip1 capacity for direct binding to PCNA and suppression of CDK complexes. Double immunohistochemical examination for 8-OHdG and apoptosis clearly indicated that the induction of p21WAF1/Cip1 might be related to the oxidative DNA damage, which occur in pericentrally localized hepatocytes. Biochemical and molecular biological data of this study corresponded to the results of our immunohistochemical analysis. However, the exact mechanisms deserve further investigations.
Recently, an increase of bromodeoxyuridine (BrdU) labeling indices, 1 week post-treatment with 0.05% PB, was demonstrated in the livers of c-myc/TGF-
double-transgenic and C57BL/10J mice (47,48). Another short-term study, performed with AP Wistar rats, revealed enhancement of cell proliferation after 3 days of treatment, and thereafter return to the control level (49). Rise in the PCNA index in our study at day 6 post-treatment with PB is thus generally in line with the literature (Figure 4
). Recently, the generation of intracellular ROS is reported to result in the elevation of mitogen activated kinases activities and DNA synthesis, what might be related to the increase of CD1 mRNA expression and PCNA index observed in the present experiment (50).
The fact of a reduced apoptotic index at day 10 possibly suggests a link to activation of 8-OHdG repair mechanisms. On the other hand, depression of apoptosis by PB has been recently explained on the basis of its ability to inhibit p53 (51), p21WAF1/Cip1 (52) and enhance the bcl-2 gene family expression (53). Furthermore, PB may co-operate with c-myc and TGF-
in the selective inhibition of apoptosis through diverse molecular pathways (54).
In conclusion, our results indicate that application of non-genotoxic liver tumor promoter PB at a high dose to rats induces conspicuous but reversible alteration to DNA oxidative base modifications in the nuclear of hepatocytes via generation of oxidative stress and affects expression of genes involved in cell-cycle regulation with a sequence of events leading first to cell proliferation, then to cell-cycle arrest, apoptosis and finally apparent accommodation. How the treatment with non-genotoxic chemicals impacts on these in rats after initiation of hepatocarcinogenesis with genotoxic carcinogen is the subject for our further investigations.
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Notes
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3 To whom requests for reprints should be sentEmail: fukuchan{at}med.osaka-cu.ac.jp 
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Acknowledgments
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We thank Emi Kawakami, Miyoko Yamanaka and Atsuko Tominaga for their technical assistance, and Mari Dokoh and Akiko Wakamiya for their help during preparation of this manuscript. This research was supported by a grant from the Japan Science and Technology Corporation, included into the Project of Core Research for Evolutional Science and Technology (CREST) in Japan.
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Received February 13, 2001;
revised August 25, 2001;
accepted October 29, 2001.