Department of Pathology, Medical College of Ohio, Toledo, Ohio 43614-5806
Received May 18, 2004; accepted August 4, 2004
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ABSTRACT |
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Key Words: dibromoacetic acid; DNA hypomethylation; insulin-like growth factor 2; liver; c-myc; peroxisome proliferation.
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INTRODUCTION |
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Because much fewer toxicology and carcinogenicity data are available for DBA (Boorman, 1999), its long-term carcinogenicity is now being evaluated by the US National Toxicology Program. The carcinogenic activity of DCA and TCA appears not to result from genotoxic activity, as in vitro and in vivo tests for their genotoxicity have not demonstrated significant activity (Anna et al., 1994
; Fahrig et al., 1995
; Ferreira-Gonzalez et al., 1995
; Fox et al., 1996
; Meier and Blazak, 1990
; Schroeder et al., 1997
). The mechanism of DCA and TCA carcinogenicity has been hypothesized to be epigenetic, involving DNA methylation (Pereira et al., 2004a
; Tao et al., 2000a
, 2000b
). DNA methylation as 5-methylcytosine (MeC) is an epigenetic mechanism that regulates chromosomal stability, histone acetylation, and the expression of genes (Eden et al., 2003
; Feinberg and Tycko, 2004
; Momparler, 2003
; Razin and Shemer, 1999
). DNA hypomethylation is a common early event in human and animal tumors that has been proposed to be a key factor in expanding the population of cells at risk for developing cancer (Baylin et al., 1998
; Dunn, 2003
). In mouse liver and hepatic tumors, we have reported that DCA and TCA caused global hypomethylation of DNA, as well as hypomethylation in the promoter regions of the insulin-like growth factor 2 (IGF-II), c-jun, and c-myc protooncogenes (Tao et al., 1998
, 2000a
, 2000b
, 2004
). We choose to evaluate the methylation of two of these genes, IGF-II and c-myc, because the expression of their mRNA is increased in mouse liver tumors. Other nongenotoxic mouse liver carcinogens, including trichloroethylene, 2,4-dichlorophenoxyacetic acid, dibutyl phthalate, gemfibrozil, Wyeth-14,643, and phenobarbital, have been shown to decrease the methylation of DNA (Counts et al., 1996
, 1997
; Ge et al., 2001
, 2002
; Tao et al., 1999
). Thus, the ability of many nongenotoxic carcinogens to cause DNA hypomethylation suggests that it might be a common mechanism for their carcinogenic activity and could be useful as a biomarker to identify other nongenotoxic liver carcinogens.
Although DCA and TCA are structurally similar, differing by only one chlorine atom, their biochemical toxicology and histopathology have been shown to be quite different (Bull et al., 1990; Latendresse and Pereira, 1997
; Pereira, 1996
; Pereira and Phelps, 1996
; Pereira et al., 1997
, 2001
; Tao et al., 1996
;). For example, in female B6C3F1 mice, liver tumors induced by DCA were predominantly eosinophilic and contained glutathione S-transferase-
(GST-
), whereas those induced by TCA were basophilic and lacked GST-
(Latendresse and Pereira, 1997
; Pereira, 1996
). Dichloroacetic acidtreated mice displayed greatly enlarged livers characterized by a marked cytomegaly and massive accumulations of glycogen in hepatocytes, while TCA caused a smaller increase in liver size without significant cytomegaly and glycogen accumulation (Bull et al., 1990
). Both DCA and TCA are peroxisome proliferators; however, prolonged exposure to TCA, but not to DCA, resulted in maintenance of the increased activity of the peroxisomal enzyme, acyl-CoA oxidase (DeAngelo et al., 1989
; Goldsworthy and Popp, 1987
; Parrish et al., 1996
). In the present study, we evaluated in the liver of female B6C3F1 mice and male F-344 rats the ability of DBA to cause (1) hypomethylation of DNA and the IGF-II and c-myc genes, (2) accumulation of glycogen, and (3) enhancement of peroxisome proliferation. Our goal was to determine whether the molecular and biochemical alterations induced by DCA and TCA were shared by DBA.
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MATERIALS AND METHODS |
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Determination of the methylation of DNA and of the c-myc and IGF-II Genes. DNA was isolated from the liver by digestion with 400 µg/ml RNase A and 200 µg/ml proteinase K followed by organic extraction with phenol, chloroform, and isoamyl alcohol. Global methylation of DNA was determined by dot-blot analysis using a monoclonal antibody specifically against 5-methylcytosine, as described previously (Tao et al., 2004). DNA was dotted onto a Hybond nitrocellulose membrane. The membrane was then probed with a 1:1,000 dilution of mouse monoclonal primary antibody specific against 5-MeC (Eurogentec Company, Belgium), washed with T-BST (Tris-buffered saline plus Tween 20, pH7.6) and incubated with 1:2,000 dilution of horseradish peroxidase (HRP)conjugated secondary anti-mouse-IgG antibody for 1 h. The membrane was then treated with enhanced-chemiluminescence Western blotting detection reagents and exposed to Kodak autoradiograph films. The optical density (OD) for the 5-MeC in DNA was measured with the Scion Image Analysis System (Scion Corp., Frederick, MD). Equal loading of the DNA was indicated by equal intensity of 0.02% methylene blue staining.
Methylation in the promoter region of the mouse c-myc gene was determined by digestion with the methylation-sensitive restriction endonuclease Hpa II followed by Southern blot analysis (Tao et al., 2000a 2000b
). The digested DNA was electrophoresed on 1% agarose gel and transferred by vacuum to Hybond-N+ nylon membranes. The membranes were hybridized with a random 32P-labeled c-myc probe. Autoradiography was performed with Kodak Biomax MR X-ray film.
Methylation of CpG sites between exons 4 and 5 in the differentially methylated region-2 (DMR-2) of the mouse IGF-II gene was determined by a bisulfite-modified DNA sequencing procedure (Tao et al., 2004). The DNA was treated with sodium bisulfite to convert unmethylated cytosine to uracil, while methylated cytosine was resistant. Polymerase chain reaction amplification was performed with the following primers: upstream, 5'-TTG ATG GTA TTA TAT TGT AGA ATT AT-3' and downstream, 5'-AAC TAA AAT TAT CTA TCC TAT AAA AC-3' (Hemberger et al., 1998
; Olek et al., 1996
; Tao et al., 2004
). The PCR-generated DNA fragments (616 bp) were ligated into the TA cloning vector, pCR 2.1 and transformed into One Shot TOP10F' Chemically Competent Escherichia coli cells according to standard protocols (Invitrogen, Carlsbad, CA). Plasmid DNA was automatically sequenced with an ABI Model 310 DNA sequencer (PerkinElmer, Norwalk, CT).
RT-PCR analysis for the mRNA expression of the c-myc and IGF-II genes. mRNA expression of the c-myc and IGF-II genes was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) as described previously (Tao et al., 2004). Total RNA was extracted from liver tissues using TRIzol Reagent and treated with DNase I. cDNA was synthesized from 1 µg of total RNA using avian myeloblastosis virus reverse transcriptase. The c-myc and IGF-II genes were respectively co-amplified with glyceraldehyde 3-phosphate dehydrogenase (Gapdh) used as an internal standard. Primer sequences for the c-myc gene (GenBank Database Accession Number: Z38066) were upstream: 5'-CTA CTC GCC TCC CTG CGA AG-3' (nt 318337 bp) and downstream: 5'-GCG TAG CAG GCT CCA CGT CG-3' (nt 588607 bp); for the IGF-II gene (GenBank Database Accession Number: NM_010514) were upstream: 5'-GGC CCC GGA GAG ACT CTG TGC-3' (nt 10141034 bp) and downstream: 5'-GAA GTC GTC CGG AAG TAC GG-3' (nt 12311250 bp); and for the Gapdh gene (GenBank Database Accession Number: NM_008084) were upstream: 5'-ATG GTG AAG GTC GGT GTG AAC G-3' (nt 4768 bp) and downstream: 5'-GTT GTC ATG GAT GAC CTT GGC C-3' (nt 520541 bp). The PCR products were electrophoresed and the optical density of the products was measured with the Scion Image Analysis System.
Determination of liver glycogen. Glycogen was isolated from 3050 mg of liver by dissolving the tissue in 30% potassium hydroxide saturated with Na2SO4 for 30 min at 100°C, followed by ethanol precipitation. Glycogen content was determined by the phenol-sulfuric acid spectrophotometric method, as described previously (Pereira et al., 2004a) and expressed as micrograms of glycogen per milligram of liver.
Determination of peroxisomal lauroyl-CoA oxidase activity. Peroxisomal acyl-CoA oxidase activity was determined using lauroyl-CoA as the substrate and the fluorometric method based on hydrogen peroxide oxidation of 4-hydroxyphenylacetic acid (Kvannes and Flatmark, 1991; Poosch and Yamazaki, 1986
). Liver was homogenized in 60 mM potassium phosphate (pH 7.0) containing 0.2% Triton X-100 and centrifuged at 1,000 x g. The 1,000 x g supernatant was diluted to 40 mg protein/ml and a 0.5 ml aliquot was added to 0.5 ml of a reaction mixture containing 0.2% Triton X-100, 60 mM potassium phosphate (pH 7.0), 1 mM 4-hydroxyphenylacetic acid, 4 U/ml HRP, 20 mM FAD, and 60 mM lauroyl-CoA. The reaction was incubated at 37°C for 30 min in a shaking water bath and then stopped by the addition of 2 mM KCN in 100 mM sodium bicarbonate, pH 10.5. Fluorescence was determined using excitation at 320 nm and emission at 405 nm. Peroxisomal lauroyl-CoA oxidase activity was expressed as nanomoles of hydrogen peroxide produced/min/mg protein.
Statistical analysis. The results were analyzed for statistical significance by one-way analysis of variance followed by the Bonferroni t-test. Statistical significance was indicated by a p value < 0.05 unless otherwise indicated.
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RESULTS |
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DISCUSSION |
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DNA hypomethylation has been shown to release methyl binding proteins and associated histone deacetylase that increases the acetylation of histones and opens up the chromatin to allow the binding of transcription factors and RNA polymerase to the promoter sequences of the target genes, resulting in increased expression of the genes (Dunn, 2003; Eden et al., 2003
; Feinberg and Tycko, 2004
; Momparler, 2003
). DNA hypomethylation has also been proposed to promote the hypermethylation of tumor suppressor genes resulting in their downregulation (Dunn, 2003
). The increased expression of protooncogenes and the downregulation of tumor suppressor genes are expected to result in a growth advantage for the affected cells that enhances the risk of cancer. This has led to DNA hypomethylation being proposed as a factor in the carcinogenic mechanism of nongenotoxic liver carcinogens (Counts and Goodman, 1995
; Pereira et al., 2004a
, 2004b
; Tao et al., 2000a
, 2004
). It was further proposed that the ability to induce DNA hypomethylation could be used to screen chemicals for nongenotoxic carcinogenic activity.
Dibromoacetic acid induced hypomethylation of DNA, and of the promoter regions of the c-myc and IGF-II genes. DBA also increased the mRNA expression of the two genes that was associated with their hypomethylation. Dichloroacetic acid and TCA also induced hypomethylation of DNA and of the two genes, and increased the expression of the genes (Tao et al., 1998, 2000a
, 2000b
, 2004a). When administered at concentrations similar to those of the DBA used in our study, DCA and TCA have been shown to be mouse liver carcinogens (Bull et al., 1990
; Herren-Freund et al., 1987
; Pereira, 1996
; Pereira and Phelps, 1996
). Hence, using the concentrations of DCA and TCA that were high relative to their concentration found in drinking water, these studies identified a potential carcinogenic hazard for the two chloroacetic acids (IARC, 1995
). The ability of DBA to induce hypomethylation of DNA, c-myc, and IGF-II would suggest that it is also a nongenotoxic liver carcinogen in mice, albeit at the concentrations higher than those found in drinking water.
The two haloacetic acids, DCA and TCA, appeared to elicit different histopathology (Bull et al., 1990; Herren-Freund et al., 1987
; Latendresse and Pereira, 1997
; Pereira, 1996
; Pereira and Phelps, 1996
). Dichloroacetic acid, but not TCA, induces glycogen accumulation in mouse liver. Dibromoacetic acid also induces glycogen accumulation in both mouse and rat liver, suggesting that its activity might be more similar to DCA than to TCA. Both DCA and TCA induce peroxisome proliferation in rodent liver (DeAngelo et al., 1989
; Goldsworthy and Popp, 1987
; Parrish et al., 1996
), and we report here that DBA also induces peroxisome proliferation in mouse and rat liver. Liver tumors induced by TCA have been demonstrated to be basophilic staining, similar to those induced and promoted by other peroxisome proliferators. In contrast, tumors induced by DCA, especially in female mice, have been demonstrated to be eosinophilic staining. Should DBA prove to be a liver carcinogen, the associated DNA hypomethylation as well as the associated increased expression of protooncogenes, glycogen accumulation, and peroxisome proliferation could be involved in the carcinogenic process. Furthermore, these associated biochemical and molecular characteristics could be used to distinguish whether the development and histopathology of the tumors are more similar to those induced by DCA or TCA.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Division of Hematology and Oncology, Department of Internal Medicine, College of Medicine and Public Health, The Ohio State University, 300 West Tenth Avenue, 1148 CHRI, Columbus, OH 43210; Fax: (614)-293-4072. E-mail: tao-2{at}medctr.osu.edu.
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