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Carcinogenesis Advance Access originally published online on January 19, 2008
Carcinogenesis 2008 29(3):638-646; doi:10.1093/carcin/bgm303
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Genetic and epigenetic changes in rat preneoplastic liver tissue induced by 2-acetylaminofluorene

Tetyana V. Bagnyukova{dagger}, Volodymyr P. Tryndyak{dagger}, Beverly Montgomery, Mona I. Churchwell, Adam R. Karpf1, Smitha R. James1, Levan Muskhelishvili2, Frederick A. Beland and Igor P. Pogribny*

Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA
1 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
2 Toxicologic Pathology Associates, National Center for Toxicological Research, Jefferson, AR 72079, USA

* To whom correspondence should be addressed. Tel: +1 870 543 7096; Fax: +1 870 543 7720;Email: igor.pogribny{at}fda.hhs.gov


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Genotoxic carcinogens, including 2-acetylaminofluorene (2-AAF), in addition to exerting their genotoxic effects, often cause a variety of non-genotoxic alterations in cells. It is believed that these non-genotoxic effects may be indispensable events in tumorigenesis; however, there is insufficient knowledge to clarify the role of carcinogens in both the genetic and epigenetic changes in premalignant tissues and a lack of conclusive information on the link between epigenetic alterations and carcinogenic exposure. In the current study, we investigated whether or not the mechanism of 2-AAF-induced hepatocarcinogenesis consists of both genotoxic (genetic) and non-genotoxic (epigenetic) alterations. Male and female Sprague–Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 6, 12, 18 or 24 weeks. The levels of DNA adducts obtained from 2-AAF in liver and kidney tissues were assessed by high-performance liquid chromatography combined with electrospray tandem mass spectrometry (HPLC-ES-MS/MS). N-(Deoxyguanosine-8-yl)-2-aminofluorene was the major adduct detected at all time points in both tissues. Global DNA methylation in the livers and kidneys, as determined by an HpaII-based cytosine extension assay and by HPLC-ES-MS/MS, did not change over the 24-week period. In the livers of male rats, there was a progressive decrease of global and long interspersed nucleotide element-1-associated histone H4 lysine 20 trimethylation, as well as hypermethylation of the p16INK4A gene. These epigenetic changes were not observed in the livers of female rats or the kidneys of both sexes. Importantly, morphological evidence of formation and progression of neoplastic process was observed in the liver of male rats only. In conclusion, we have demonstrated that exposure of rats to genotoxic hepatocarcinogen 2-AAF, in addition to formation of 2-AAF-specific DNA lesions, resulted in substantial alterations in cellular epigenetic status.

Abbreviations: 2-AAF, 2-acetylaminofluorene; dG-C8-AF, N-(deoxyguanosine-8-yl)-2-aminofluorene; DNMT1, DNA methyltransferase 1; GST-P, glutathione-S-transferase placental form; HPLC-ES-MS/MS, high-performance liquid chromatography combined with electrospray tandem mass spectrometry; H4K20me3, histone H4 lysine 20 trimethylation; H3K9me3, histone H3 lysine 9 trimethylation; LINE-1, long interspersed nucleotide element-1; ORF1, open reading frame 1; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; ROP dG-C8-AF, oxidation products of N-(deoxyguanosine-8-yl)-2-aminofluorene


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
The aromatic amine 2-acetylaminofluorene (2-AAF) is a genotoxic carcinogen that induces tumors in a variety of species and tissues (1–3). In male rats, 2-AAF is a powerful complete liver carcinogen leading to tumor formation without any additional intervention (4,5). It is widely believed that the covalent interaction of metabolic derivatives of 2-AAF with DNA is a critical step in the initiation of tumorigenesis (3); however, it has also been suggested that although the presence of DNA adducts per se is a necessary prerequisite for tumor initiation, it is not sufficient for tumor formation (4–6), which results from much broader alterations in cellular homeostasis, mainly from the inability of cells to maintain and control properly the expression of genetic information. Furthermore, tissue-specific or sex-specific tumor development cannot be explained only by DNA adduct formation (4).

Genotoxic carcinogens, including 2-AAF, in addition to exerting their genotoxic effects, often cause a variety of non-genotoxic alterations in cells (4,5,7,8). These non-genotoxic effects, especially the ability of 2-AAF to induce chronic liver toxicity and disturb the biochemical and physiological cellular homeostasis, may play an important role in the generation of tumors induced by 2-AAF (5,7,8). Emerging evidence suggests that carcinogen-induced non-genotoxic changes, especially alterations in the cellular epigenetic status (9–11), maintained by a sustained stress environment, result in the emergence of epigenetically reprogrammed cells. These epigenetically reprogrammed cells are characterized by epigenetic alterations similar to those frequently found in tumor cells, such as global DNA hypomethylation, changes in histone modification patterns, hypomethylation of proto-oncogenes and DNA repetitive sequences, and hypermethylation of tumor suppressor genes, especially genes involved in the cell-cycle control and apoptosis (12). It is believed that the appearance of epigenetically reprogrammed cells is an indispensable event in the origin of cancer (10–12); however, there is insufficient knowledge to clarify the role of carcinogens in both genetic and epigenetic changes in premalignant tissues and a lack of conclusive information on the link between epigenetic alterations and carcinogenic exposure (13).

Based on these considerations, the goals of this study were (i) to elucidate the underlying mechanisms of 2-AAF-induced rat hepatocarcinogenesis, especially to define whether or not the exposure to 2-AAF, in addition to specific carcinogen-induced genotoxic changes, is characterized by disruption of cellular epigenetic status and (ii) to determine the role of 2-AAF-induced epigenetic alterations in the mechanism of chemically induced liver carcinogenesis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Chemicals
N-(deoxyguanosine-8-yl)-2-aminofluorene (dG-C8-AF) was synthesized by reaction N-acetoxy-2-trifluoroacetylaminofluorene with deoxyguanosine using the method of Lee et al. (14). dG-C8-AAF was prepared by reacting N-acetoxy-2-AAF with deoxyguanosine as outlined by Kriek et al. (15). Oxidation products of N-(deoxyguanosine-8-yl)-2-aminofluorene (ROP dG-C8-AF) were prepared by treating dG-C8-AAF with 1 N NaOH (16). [15N5]dG-C8-AF, [15N5]dG-C8-AAF and [15N5]ROP dG-C8-AF were synthesized by substituting [15N5]deoxyguanosine for deoxyguanosine. Stock solutions of the DNA adducts were prepared in methanol and their concentrations determined by using the molar extinction coefficients reported by Kriek et al. (15). The concentrations of ROP dG-C8-AF and [15N5]ROP dG-C8-AF were estimated by assuming quantitative conversion of dG-C8-AAF and [15N5]dG-C8-AAF to ROP dG-C8-AF and [15N5]ROP dG-C8-AF upon treatment with base.

Animals, treatment and tissue preparation
Weaning male and female Sprague–Dawley rats were obtained from the National Center for Toxicological Research breeding facility, housed in a temperature-controlled (24°C) room with a 12-h light–dark cycle and given ad libitum access to water and NIH-31 laboratory diet. At 6 weeks of age, the rats (mean body weight 150 g) were allocated randomly to receive either NIH-31 diet containing 0.02% of 2-AAF or control NIH-31 diet. Diets were stored at 4°C and given ad libitum, with twice a week replacement. Four rats per diet group and three rats per control group were killed at 6, 12 and 18 weeks and six rats per diet group were killed at 24 weeks after initiation of the 2-AAF diet. The livers, kidneys and spleens were excised, frozen immediately in liquid nitrogen and stored at –80°C for subsequent analysis. All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the National Center for Toxicological Research.

Isolation and hydrolysis of liver and kidney DNA from rats
Genomic DNA was isolated from rat liver and kidney tissues by standard digestion with proteinase K, followed by phenol–chloroform extraction and ethanol precipitation (17). DNA samples (typically 100 µg) were enzymatically hydrolyzed to nucleosides for subsequent DNA adduct analyses by high-performance liquid chromatography combined with electrospray tandem mass spectrometry (HPLC-ES-MS/MS).

HPLC-ES-MS/MS analyses of DNA adducts
HPLC-ES-MS/MS analyses were performed with a liquid handling system consisting of an Alliance 2795 Separations module, a Dionex GP40 quaternary gradient pump and an automated switching valve (TPMV, Rheodyne, Rohnert Park, CA). The Alliance 2795 system was used for sample injection, sample concentration and regeneration of the trap column. The Dionex pump was used to backflush the trap column to the analytical column during analysis and to keep a constant flow of mobile phase through the analytical column into the mass spectrometer during the sample loading and preparation periods.

Each sample was loaded onto a reverse-phase trap column [Luna C18(2), 2 x 30 mm, 3 µm] with 95% aqueous acetonitrile at a flow rate of 0.2 ml/min for 1.5 min. Polar components were washed to waste for 2 min with 95% aqueous acetonitrile at a flow rate of 0.5 ml/min. After switching the divert valve, the concentrated sample was backflushed from the trap column onto the analytical column [Luna C18(2), 2 x 150 mm, 3 µm] with a linear gradient of 30–45% aqueous acetonitrile over 10 min, and the sample content was eluted into the mass spectrometer. After 3 min, the valve was switched back to the load position and the trap column was washed (to waste) with 50% aqueous acetonitrile at a flow rate of 0.5 ml/min. The trap column was then equilibrated with the starting mobile phase 95% aqueous acetonitrile. The total run time for sample preparation and analysis was 19.6 min.

A Quattro Ultima quadrupole mass spectrometer, equipped with an electrospray interface, was used with a source block of 100°C and a desolvation temperature of 350°C. Nitrogen was the desolvation (750 l/h), cone (300 l/h) and nebulizing gas. Argon was the collision gas, at a collision cell pressure of 2.0 x 10–3 Mbar. Positive ions were acquired in the multiple reaction-monitoring mode (dwell time of 0.1 s and interchannel delay of 0.03 s). The following transitions were monitored: for dG-C8-AF, the protonated molecule [(M + H)+] (m/z 447) to [BH2]+ (m/z 331) and (m/z 447) to (m/z 314); for [15N5]dG-C8-AF, [(M + H)+] (m/z 452) to [BH2]+ (m/z 336); for dG-C8-AAF, [(M + H)+] (m/z 489) to [BH2]+ (m/z 373) and (m/z 373) to (m/z 331); for [15N5]dG-C8-AAF, [(M + H)+] (m/z 494) to [BH2]+ (m/z 378) and (m/z 378) to (m/z 336); for ROP dG-C8-AF, [(M + H)+] (m/z 463) to [BH2]+ (m/z 347) and (m/z 463) to (m/z 277) and for [15N5]ROP dG-C8-AF, [(M + H)+] (m/z 468) to [BH2]+ (m/z 352). The cone voltage was 35 V and the collision energy was 25 eV for all transitions except for the dG-C8-AAF [M + H]+ to [BH2]+ transitions, which was 15 eV, and the dG-C8-AF and ROP dG-C8-AF confirmation transitions, which were at 38 and 35 eV, respectively. Samples were quantified by comparing the peak areas of dG-C8-AF, dG-C8-AAF and ROP dG-C8-AF with the respective [15N5] internal standards. The levels of dG-C8-AF in liver and kidney DNA were determined using 25 pg of [15N5]dG-C8-AF and 1 µg of DNA. The levels of dG-C8-AAF and ROP dG-C8-AF were measured using 25 pg of [15N5]dG-C8-AAF and 25 pg of [15N5]ROP dG-C8-AF and 95 µg of DNA.

Determination of global DNA methylation status by cytosine extension assay
The extent of global DNA methylation was evaluated with a radiolabeled [3H]dCTP extension assay as described in Pogribny et al. (18).

Determination of global DNA methylation status by quantification of 5-methyl-2'-deoxycytidine level
The levels of 5-methyl-2'-deoxycytidine in hepatic DNA was determined by HPLC-ES-MS/MS as indicated in Song et al. (19).

Methylation analysis of long interspersed nucleotide element-1
The methylation status of long interspersed nucleotide element-1 (LINE-1) was determined by a combined bisulfite restrivtion analysis assay, which consists of a standard bisulfite modification of genomic DNA, subsequent polymerase chain reaction (PCR) amplification and digestion of the PCR product with the appropriate restriction endonuclease as reported previously in detail (20).

Determination of the p16INK4A methylation status by methylation-specific PCR
The methylation status of the CpG island located within promoter and first exon of the p16INK4A gene was determined by methylation-specific PCR analysis (21).

Analysis of histone H3 and histone H4 modifications
The status of histone H3 lysine 9 trimethylation (H3K9me3) and histone H4 lysine 20 trimethylation (H4K20me3) in the livers of control rats and rats fed 2-AAF was determined by western blot analysis as described (22).

Western blot analysis of protein expression
The protein levels of DNA methyltransferase 1 (DNMT1), proliferating cell nuclear antigen (PCNA) and β-actin in the livers of control rats and rats fed 2-AAF was determined as described previously (20).

Quantitative real-time PCR analysis
Total RNAs were isolated from the liver tissue using TRI Reagent (Ambion, Austin, TX) according to the manufacturer’s instruction. To prevent genomic DNA contamination, all RNA samples were subjected to DNase I digestion. The quality of the total RNA was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). cDNAs were synthesized with Moloney murine leukemia virus reverse transcriptase (GE Healthcare Biosciences, Piscataway, NJ) using 5 µg of total RNA, which had been primed with oligo(dT), as the template. Two-step quantitative real-time PCR was performed for measurement of LINE-1 open reading frame 1 (ORF1) and p16INK4A expression using a SYBR GreenER SuperMix (Invitrogen, Carlsbad, CA) for iCycler (Bio-Rad, Hercules, CA) with 50 cycles of 1 min at 95°C and 1.5 min at 60°C. Fluorescence was measured at 60°C after each cycle using an iCycler apparatus (Bio-Rad). After the final cycle, melting curve analysis of all samples was conducted within the range from 55–95°C. Relative quantification of gene expression was performed by using glyceraldehyde-3-phosphate dehydrogenase as an internal control. The threshold cycle and the Formula method were used for calculating the relative amount of the target RNA. Quantitative real-time PCR was performed at least three times and always included a no-template sample as a negative control.

Chromatin immunoprecipitation assay
Chromatin immunoprecipitation assay for LINE-1-associated histone methylation with primary antibodies against H3K9me3 and H4K20me3 (Upstate, Charlottesville, VA) was performed as described previously (23).

Immunohistochemistry
The status of glutathione-S-transferase placental form (GST-P) expression in the livers of male and female rats was determined after feeding the rats with 2-AAF-containing diet for 24 weeks. Formalin-fixed paraffin-embedded liver sections were deparaffinized and rehydrated. Endogenous peroxidase was inhibited by incubating with freshly prepared 3% hydrogen peroxide containing 0.1% sodium azide for 10 min at room temperature. Non-specific staining was blocked with normal goat 10% serum (Sigma, St Louis, MO) for 20 min at room temperature. The sections were then incubated with rabbit anti-human GST-P antibody (DAKO, Carpinteria, CA) at the dilution of 1:100 (10 µg/ml) for 1 h at room temperature. After incubation with primary antibody, tissue sections were incubated with biotinylated goat anti-rabbit IgG (ExtrAvidin Kit, Sigma) at a dilution of 1:30 for 30 min at room temperature, and then with streptavidin-conjugated horseradish peroxidase (ExtrAvidin Kit, Sigma) at the dilution of 1:30 for 30 min at room temperature. Staining was developed with diaminobenzidine (Sigma) for 5 min at room temperature, and sections were counterstained with hematoxylin and mounted with Permount (Fisher Scientific, Pittsburgh, PA). All sections were examined by light microscopy (BX40 Olympus, Tokyo, Japan).

Virtual microscopy and image analysis
GST-P-stained rat liver sections were scanned and digital images were obtained with Aperio Scanscope System (Aperio Technologies, Vista, CA). The proportion of the area that stained positive for GST-P in the digital images was evaluated with Positive Pixel Count Algorithm (Aperio Technologies).

Statistical analyses
Results are presented as mean ± SD. Statistical analyses were conducted by two-way analysis of variance, with post hoc pairwise comparisons being performed by Student–Newman–Keuls test. P values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Hepatic 2-AAF–DNA adduct levels
HPLC-ES-MS/MS was used to assess the levels of dG-C8-AF, dG-C8-AAF and ROP dG-C8-AF in hepatic and kidney DNA of male and female rats fed 2-AAF and corresponding control rats. Multiple reaction-monitoring chromatograms of hepatic DNA from a male rat fed 2-AAF for 12 weeks are shown in supplementary Figure 1, available at Carcinogenesis Online. Hepatic DNA adduct levels were measured at 6, 12, 18 and 24 weeks of 2-AAF feeding; measurements of kidney DNA adduct levels were made at 12 and 24 weeks. The results are shown in Figure 1. In male rats, the levels of dG-C8-AF at 6 and 12 weeks in liver DNA were significantly greater than the levels at 18 and 24 weeks. In female rats, the levels of dG-C8-AF were relatively constant at each time point, with the values being significantly less than in the male rats at 6 and 12 weeks, but significantly greater than in the male rats at 18 and 24 weeks. The levels of dG-C8-AF in kidney DNA were similar in both sexes at each time point. In male rats, the levels of dG-C8-AF were higher in liver DNA than in kidney DNA when measured at 12 weeks, whereas the opposite relationship was observed at 24 weeks. In female rats, the level of dG-C8-AF was higher in liver DNA compared with kidney DNA at both 12 and 24 weeks.


Figure 1
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  		Fig. 1. Levels of dG-C8-AF in the livers of male (A) and female (B) rats fed 2-AAF or control diet for 6, 12, 18 or 24 weeks and in the kidneys of male (C) and female (D) rats fed 2-AAF or control diet for 12 or 24 weeks. *Significantly different from control at the same time point.

 
Low levels of dG-C8-AAF (0.03–0.23 adducts/106 nucleotides) were found in male rat liver DNA, with the highest values being detected at 6 and 12 weeks of 2-AAF feeding. Even lower levels of dG-C8-AAF (<0.005–0.04 adducts/106 nucleotides) were found in female rat liver DNA. dG-C8-AAF was present in male kidney DNA (0.02–0.05 adducts/106 nucleotides) but was not detected (<0.005 adducts/106 nucleotides) in female kidney DNA. ROP dG-C8-AF was found in male (0.03–1.05 adducts/106 nucleotides) and female (0.05–0.89 adducts/106 nucleotides) liver DNA and male (0.04–0.21 adducts/106 nucleotides) and female (0.04–0.12 adducts/106 nucleotides) kidney DNA.

Effect of 2-AAF on DNA methylation in liver and kidney tissues
The presence of DNA adducts induced by genotoxic carcinogens, including 2-AAF, may perturb the DNA methylation status, which could be significant for cancer induction (9–11). In order to examine whether or not accumulation of 2-AAF–DNA adducts is associated with aberrant global DNA methylation, we first assessed the methylation status with a sensitive cytosine extension assay that measures proportion of unmethylated CpG sites in DNA (18). Figure 2 shows that the extent of DNA methylation in the livers of male (panel A) and female (panel B) rats fed 2-AAF-containing diet did not change over 24 week period and did not differ between the 2-AAF-exposed and age-matched control rats. In contrast, the level of DNA hypomethylation in the kidneys of 2-AAF-treated male rats (panel C) increased by 29 and 55%, respectively, after 18 and 24 weeks of exposure compared with age-matched control rats. Treatment with 2-AAF did not change the extent of DNA methylation in the kidneys of female rats (panel D) or spleens of male and female rats (data not shown).


Figure 2
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  		Fig. 2. Effect of 2-AAF on the level of DNA methylation. DNA methylation in liver of male (A) and female (B) and kidneys of male (C) and female (D) rats fed 2-AAF and their respective age-matched control rats was measured by the cytosine extension assay after pretreating the DNA with methylation-sensitive restriction endonuclease HpaII, which cleaves CCGG sequences when internal cytosine residues are unmethylated on both strands. The extent of [3H]dCTP incorporation is directly proportional to the number of unmethylated CCGG sites. *Significantly different from control at the same time point.

 
To assess further the effect of 2-AAF exposure on global DNA methylation, HPLC-ES-MS/MS analyses (19) were conducted to determine the levels of 5-methyl-2'-deoxycytidine in DNA isolated from the liver of male rats fed 2-AAF and from control rats. The level of 5-methyl-2'-deoxycytidine in livers did not differ between the 2-AAF-exposed and age-matched control rats at any time point (supplementary Table 1, available at Carcinogenesis online).

A recent study has shown that the methylation pattern of mammalian genome consists of short unmethylated domains (<4 kb) embedded within a matrix of long methylated domains of large number of repetitive DNA sequences (24). Additionally, it has been proposed that one of the key functions of DNA methylation in normal somatic cells is to maintain the stability of the genome by silencing the expression of these repetitive sequences (25,26). In a previous study, using a tamoxifen-induced model of genotoxic rat hepatocarcinogenesis, we showed that feeding female Fisher 344 rats a tamoxifen-containing diet leads to a decrease of LINE-1 methylation in liver (20). In view of this, we measured the methylation status of LINE-1 in the livers of male and female rats exposed to 2-AAF and in the livers of age-matched control rats. Feeding 2-AAF did not change the LINE-1 methylation in liver of male and female rats at any time interval (data not shown).

Effect of 2-AAF on the expression of DNMT1 and PCNA in liver
DNMT1 is the main cellular enzyme responsible for maintaining DNA methylation patterns in somatic mammalian cells. Additionally, there is a well-established link between expression of DNA methyltransferases and DNA replication (27). Therefore, we assessed whether or not 2-AAF exposure was associated with an altered level of DNMT1 and PCNA proteins in liver of male and female rats. Figure 3 shows that feeding male rats with 2-AAF-containing diet for 24 weeks resulted in progressive and prominent increase in level of DNMT1 protein (panel B), whereas the level of PCNA increased slightly (panel C). In female rats, the level of DNMT1 protein in liver did not change significantly at any time points (data not shown).


Figure 3
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  		Fig. 3. Expression of DNMT1 and PCNA in the livers of control male rats and rats fed 2-AAF. Liver tissue lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to western immunoblotting using specific antibodies against DNMT1 and PCNA. Equal sample loading was confirmed by immunostaining against β-actin. These results were reproduced in two independent experiments. (A) Representative western immunoblot images are shown. (B) Quantitative analysis of DNMT1 and (C) PCNA protein levels. Data are presented as relative to age-matched control rats. Control values at each time point were considered as 100%. The histograms in each of the panels are the mean ± SD *Significantly different from the control at the same time point.

 
Status of H3K9 and H4K20 trimethylation in liver and kidney tissues
Considering the results of our previous studies showing that aberrant histone modifications play an important role in rat liver carcinogenesis induced by tamoxifen- and methyl-deficient diets (22,23), we examined alterations in the trimethylation status of histones H3K9 and H4K20 in livers of rats exposed to 2-AAF. Figure 4A shows a rapid and progressive decrease of H4K20me3 in the liver of male rats fed 2-AAF compared with age-matched control rats. After 12, 18 and 24 weeks, the levels of histone H4K20me3 in the livers of the rats fed 2-AAF was decreased by 37, 49 and 52%, respectively, compared with their age-matched controls. In contrast, the extent of H3K9me3 in the livers of male rats exposed to 2-AAF diet remained unchanged over 24 weeks (Figure 4C). Feeding 2-AAF did not change the levels of H4K20me3 (Figure 4B) and H3K9me3 (Figure 4D) in the livers of female rats at any time interval. Likewise, the levels of H4K20me3 or H3K9me3 were not altered in the kidneys of either sex (data not shown).


Figure 4
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  		Fig. 4. Western blot analysis of histone H4K20me3 and H3K9me3 in the livers of rats fed 2-AAF. Acid extracts of total histones were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to immunoblotting using specific antibodies against histone H3K9me3 and histone H4K20me3. Results are presented as change in methylation relative to control rats. Equal sample loading was confirmed by immunostaining against histone H3 and histone H4. Representative immunoblot images are shown. (A) Level of H4K20me3 in liver of male rats; (B) level of H4K20me3 in liver of female rats; (C) level of H3K9me3 in liver of male rats and (D) Level of H3K9me3 in liver of female rats. *Significantly different from control at the same time point.

 
Level of LINE-1 ORF1 expression and LINE-1-associated H4K20 trimethylation in liver and kidney tissues
Table I shows the effect of feeding 2-AAF on the expression of LINE-1 ORF1 and LINE-1-associated H4K20 trimethylation in the livers of male and female rats. In the livers of 2-AAF-fed male rats, the level of the LINE-1 ORF1 mRNA was increased after 24 weeks of exposure and was associated with a 2.25-fold decrease in H4K20me3 level at the LINE-1 regulatory region as assessed by the chromatin immunoprecipitation assay. In contrast, the levels of LINE-1 ORF1 mRNA in the livers of female rats did not differ between the 2-AAF-exposed and age-matched control groups at any time interval.


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  		Table I. Quantitative real-time PCR analysis of LINE-1 ORF1 expression and chromatin immunoprecipitation assay for LINE-1-associated H4K20 trimethylation in the livers of rats exposed to 2-AAF for 12 and 24 weeks

 
Methylation of the p16INK4A in liver and kidney tissues
The observed increase in the level of DNMT1 protein in the liver of male rats and the results of a recent study showing that DNMT1 has the capacity for catalyzing de novo methylation of CpG-rich promoters in human cells (28) prompted us to investigate the promoter methylation status of the p16INK4A gene. Hypermethylation of the p16INK4A gene and the associated loss of gene transcription are observed frequently not only in advanced but also in the early stages of hepatocarcinogenesis (29). Additionally, it has been shown that p16INK4A gene is often hypermethylated in tumorous and premalignant kidney tissues (30). In view of this, we analyzed the p16INK4A promoter methylation and expression of the p16INK4A gene in liver and kidney tissues of control rats and rats fed 2-AAF. In liver (Figure 5B) and kidneys (data not shown) of control male and female rats, only unmethylated alleles were detected, whereas 100% of livers in male rats showed progressive de novo methylation (Figure 5B), which was associated with the decreased levels of the p16INK4A mRNA (Figure 5C). In contrast, we did not observe methylation changes in liver tissue of female rats fed 2-AAF (Figure 5B) or in kidneys of either sex of rats fed 2-AAF (data not shown).


Figure 5
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  		Fig. 5. Methylation-specific PCR analysis of the p16INK4A gene methylation status in the livers of control rats and rats fed 2-AAF. (A) Partial structure of the rat p16INK4A promoter region with first exon (ref. 21; GenBank access #AY145882) with location of six analyzed CpG sites (numbered). Locations of the methylation-specific PCR primers are indicated by the arrows. (B) Bisulfite-modified DNA was amplified with two sets of primers specific to unmethylated (U) and methylated (M) cytosine residues. Each set of primers contained one sense primer, which was paired with one of two different antisense primers to determine methylation of the six CpG sites within promoter and first exon of the p16INK4A gene. The primer sequences and PCR conditions were those described by Swafford et al. (21). Methylation of the p16INK4A gene was not detected by any of the methylation-specific PCR primer pairs in normal rat liver tissues or liver tissues of 2-AAF-treated female rats. Presence of PCR products after amplification with methylation-specific primers indicates progressive de novo methylation of the p16INK4A gene in liver of 2-AAF-treated male rats. (C) Quantitative real-time PCR analysis of the p16INK4A gene expression in liver of control rats and rats fed 2-AAF. (D) Immunohistochemical demonstration of GST-P-positive foci in the livers of rats fed 2-AAF for 24 weeks. (1) control liver; (2) GST-P staining in a liver section from a male rat exposed to 2-AAF and (3) GST-P staining in a liver section from a female rat exposed to 2-AAF. Original magnification x100. The proportion (%) of GST-P-stained area in the liver sections of male and female rats fed 2-AAF is shown under the sections. Data are the means ± SDs.

 
Immunohistochemistry and image analysis
The expression of GST-P was evaluated in control and 2-AAF-treated rats after 24 weeks of feeding. Accumulated evidence suggests that increased expression of GST-P is a sensitive marker for initiated cells and represents precursor lesions, which are causally related to carcinogenesis in liver (31,32). Immunohistochemical staining of liver sections of 2-AAF-fed male rats revealed large foci consisting of GST-P-positive hepatocytes (Figure 5D, panel 2). These foci appeared to be evenly distributed throughout the entire section of the liver. Only single GST-P-positive hepatocytes or isolated minifoci consisting of 10–500 positively stained cells were observed in the livers of female rats fed 2-AAF (Figure 5D, panel 3). The proportion of the area that stained positive for GST-P in the liver sections was ~10-fold greater in the male rats as compared with the female rats (40.4 and 4.3%, respectively). No GST-P-positive foci or single-positive hepatocytes were detected in the livers of age-matched control male and female rats (Figure 5D, panel 1).


   Discussion
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Classically, the development of cancer has been viewed as a progressive multistep process of transformation of normal cells into malignant cells driven by genetic alterations that include mutations in tumor suppressor genes and oncogenes and chromosomal abnormalities (33). However, at present time, it is evident that cancer is also a disease characterized by epigenetic changes (34). The results of this study show that the 2-AAF-induced hepatocarcinogenesis in rats includes both genotoxic (genetic) and non-genotoxic (epigenetic) alterations.

To investigate the genotoxic component, an HPLC-ES-MS/MS method was developed to quantify the DNA adducts obtained from 2-AAF. Previous feeding studies with 2-AAF have relied primarily on immunoassays, 32P-postlabeling assays or both for DNA adduct quantitation (35–37). Both methods show excellent sensitivity; nonetheless, each has limitations. Quantitation by 32P-postlabeling can be compromised due to incomplete DNA digestion, losses during DNA adduct enrichment and inefficient DNA adduct labeling (38). Immunoassays can be affected by cross-reactivity of the antisera or by the failure of the antisera to recognize specific DNA adducts. HPLC-ES-MS/MS has the capability of assessing multiple DNA adducts simultaneously, can yield structural information concerning the DNA adducts, and through the use of isotopically labeled DNA adduct standards can provide accurate quantitation (39).

The major adduct detected in liver and kidney of both sexes was dG-C8-AF, which corresponds to what has been observed previously in rats fed 2-AAF when the DNA adducts were assessed by immunoassays (35). After 6 and 12 weeks of 2-AAF feeding, the highest levels of dG-C8-AF were in the livers of male rats; the values then decreased substantially by 18 and 24 weeks of treatment (Figure 1). In contrast, the levels of dG-C8-AF were relatively constant in the livers of female rats and the kidneys of both sexes. The decrease in dG-C8-AF in the liver of male rats corresponded to the appearance of GST-P-positive foci (Figure 5D, panel 2). Previous studies have shown these foci to be devoid of dG-C8-AF (40), presumably due to the inability of the preneoplastic foci to metabolize 2-AAF to a reactive electrophile (41).

In addition to dG-C8-AF, low levels of dG-C8-AAF were also found, which is consistent with what has been found by immunoassays (35). Low levels of ROP dG-C8-AF were also present in both the livers and kidneys of the 2-AAF-fed rats. This type of adduct does not appear to have been observed previously in vivo but has been suggested to have mutagenic consequences (42). The HPLC-ES-MS/MS data also suggested the presence of other DNA adducts for which isotopically labeled standards presently do not exist. One of these adducts could be 3-(deoxyguanosine-N2-yl)-2-AAF, which in livers from male rats fed 2-AAF is typically found at ~10% the level of dG-C8-AF (6,37).

In addition to 2-AAF–DNA adducts in the liver of male rats exposed to 2-AAF, we found sustained epigenetic changes consisting of a progressive loss of histone H4K20 trimethylation and promoter hypermethylation of the p16INK4A gene. Importantly, the results of our study provide the first experimental evidence that the tumor-inducing capacity of 2-AAF depends on its ability to alter the cellular epigenetic status in the target tissue. Indeed, only the liver of male rats exposed to 2-AAF was characterized by both prominent genotoxic and epigenetic changes. The evolution of these changes in male livers was closely associated with formation of GST-P-positive foci, which is widely accepted as a morphological sign of neoplastic transformation (31,32). In contrast, there were no epigenetic alterations in non-target tissues, although the accumulation of 2-AAF-adducts was found in the liver of female rats and kidneys of both sexes.

We reported previously that stable DNA hypomethylation is a hallmark of rat hepatocarcinogenesis induced by methyl-deficient diet and tamoxifen (22,23). Similar observations have been found in response to variety of carcinogenic agents (9). Unexpectedly, in the present study, we did not detect changes in global DNA methylation or in methylation of repetitive DNA sequences. This might be explained by the increased levels of DNMT1 protein in liver and the fact that 2-AAF inhibits mitosis and cell proliferation of normal hepatocytes (43). This contrasts with other genotoxic and non-genotoxic carcinogens, such as tamoxifen, methyl-deficient diets and peroxisome proliferators, that induce hepatocyte proliferation (20,44), which is an obligatory requirement for induction of DNA hypomethylation. Indeed, the absence of increase in the PCNA protein level in liver supports this suggestion.

Emerging evidence suggests a crucial role of H4K20me3 in the maintenance of genomic stability (45,46). One of the primary functions of trimethylation of H4K20, along with trimethylation of H3K9 and H3K27, is the formation of constitutive heterochromatin (45,46). H4K20me3 is concentrated at centric, pericentric and telomeric chromosomal regions (45–49) and is considered to be a prominent epigenetic mark of silenced heterochromatin (45,47). Loss of H4K20 trimethylation markedly compromises genome stability, diminishes the ability of cells to maintain cell-cycle arrest and severely impairs the viability of cells (46,47). A decreased level of H4K20me3 has been shown in several forms of human cancer (50) and, recently, in preneoplastic liver tissue of rodents during genotoxic and non-genotoxic hepatocarcinogenesis (22,23). This led to a suggestion that a low level of H4K20me3 may contribute to the etiology of cancer and can be used as an indicator and diagnostic marker for neoplastic transformation and tumor growth (47). It has been shown that major and minor satellites, long terminal repeat retrotransposons and LINE-1 retrotransposons are predominantly enriched with H4K20me3 and are contained in inactive chromatin of normal somatic cells (45, 46,49). Loss of H4K20 trimethylation was associated with increased transcription of the long terminal repeat, LINE-1 and other repetitive DNA sequences (49). In light of these observations, 2-AAF-induced loss of global and LINE-1-associated trimethylation of H4K20, accompanied by increased LINE-1 expression, in the livers of male rats may compromise genomic integrity via chromatin decondensation, the induction of centromere and telomere abnormalities, chromosome segregation defects and by activation of mobile repetitive DNA elements and proto-oncogenes. This could result in a variety of genomic instability events including cis- and trans-insertional mutagenesis, unequal homologous recombination, rearrangements and segmental duplications leading to deletions and duplications (51). The causal role of these lesions, as integral part of neoplastic transformation in etiology of cancer, including liver cancer, is now commonly accepted (52).

The results of our study and previous similar findings (53–55) indicated clearly the striking gender-specific difference in the hepatocarcinogenic effect of 2-AAF. Several possible explanations exist for such sex specificity in the development of liver tumors induced by 2-AAF exposure. First, the higher endogenous estradiol level in female rats may suppress hepatocarcinogenesis (53,54). Second, the increased efficiency of DNA repair in female rats may diminish the promoting effect of 2-AAF. Third, the lower levels of H3K9me3 and H4K20me3 in the livers of control male Sprague–Dawley rats compared with those in the livers of female rats (Table II) may be a predisposing factor for the higher susceptibility of male rats to 2-AAF-induced liver carcinogenesis. This suggestion is supported by our recent findings on the lower levels of H3Kme3 in the livers of highly susceptible to liver carcinogenesis DBA/2J and C3H/HeJ mouse strains than in resistant C57BL/6J mice (56).


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  		Table II. Western blot analysis of histone H3K9me3 and H4K20me3 in the livers of control male and female rats

 
In conclusion, we have demonstrated that exposure of rats to genotoxic hepatocarcinogen 2-AAF, in addition to leading to the formation of 2-AAF-specific DNA lesions, resulted in substantial alterations in cellular epigenetic status characterized by progressive loss of global and LINE-1-specific H4K20 trimethylation and hypermethylation of the p16INK4A gene. These epigenetic changes were observed only in the liver tissue of male rats, despite the accumulation of 2-AAF-induced genetic lesions in liver of female rats, as well as in kidney tissue of male and female rats. Importantly, morphological evidence of formation and progression of neoplastic process was observed in liver of male rats only. Therefore, we hypothesize that tumor-inducing capacity of 2-AAF depends on its ability to alter cellular epigenetic status in target tissue.


   Supplementary material
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Figure 1 and Table 1 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
FDA-NCTR Intramural Research Program; Postgraduate Research Program administered by the Oak Ridge Institute for Science and Education to T.V.B. and V.P.T.


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
The views expressed in this paper do not necessarily represent those of the U.S. Food and Drug Administration.

Conflict of Interest Statement: None declared.


   References
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 Abstract
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 Discussion
 Supplementary material
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 References
 

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Received October 10, 2007; revised December 13, 2007; accepted December 20, 2007.


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