ToxSci Advance Access originally published online on March 16, 2006
Toxicological Sciences 2006 91(2):372-381; doi:10.1093/toxsci/kfj159
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Chronic Oral Exposure to Inorganic Arsenate Interferes with Methylation Status of p16INK4a and RASSF1A and Induces Lung Cancer in A/J Mice
* Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8506, Japan; and Division of Digestive and General Surgery, Niigata University Graduate School of Medical and Dental Sciences, Niigata City 951-8510, Japan
1 To whom correspondence should be addressed at Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan. Fax: +81-29-850-2892. E-mail: xing.cui{at}nies.go.jp.
Received January 25, 2006; accepted February 27, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although inorganic arsenate (iAsV) or arsenite (iAsIII) is clearly a human carcinogen, it has been difficult to produce tumors in rodents. In the present study, we orally administered iAsV to A/J mice to examine arsenic carcinogenicity in rodent. A/J mice (male, n = 120) assigned to four groups were given drinking water containing 0, 1, 10, and 100 ppm iAsV for 18 months. At the end of experiment, the complete lungs were removed and used for examining histopathology and extracting RNA and DNA. Epigenetic effects of iAsV on DNA methylation patterns of p16INK4a and RASSF1A genes were determined by methylation-specific polymerase chain reaction. Changes of p16INK4a and RASSF1A at mRNA and protein levels were examined by reverse transcriptase–polymerase chain reaction and immunohistochemistry. Arsenic was accumulated dose dependently in the lung tissues of iAsV-exposed mice. Increase in lung tumor number and lung tumor size was observed in iAsV-exposed mice compared to the control. Histopathological examination showed that the rate of poorly differentiated lung adenocarcinoma was much higher in iAsV-exposed mice than in the control. Methylation rates appeared to be higher in a dose-related tendency in lung tumors from iAsV-exposed mice compared to the control. Lower or loss of p16INK4a and RASSF1A expression was found in lung tumors from iAsV-exposed mice, compared to that in nontumor lung tissues from both control and iAsV-exposed mice, and this reduced or lost expression was in accordance with hypermethylation of the genes. In conclusion, iAsV exposure increased lung tumor incidence and multiplicity in A/J mice. Epigenetic changes of tumor suppressor genes such as p16INK4a and RASSF1A are involved in the iAsV-induced lung carcinogenesis.
Key Words: arsenic; epigenetics; DNA methylation; lung tumor; p16INK4a; RASSF1A; lung carcinoma.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although inorganic arsenate (iAsV) or arsenite (iAsIII) exposure is clearly linked to tumor development in a variety of tissues in human, such as skin, bladder, liver, and lung (Smith et al., 1992
), it has been difficult to produce tumors in rodents (Bates et al., 1992). Deficiencies in available animal models have interfered a detailed analysis of the mechanism of arsenic-induced cancer. Recently, Waalkes et al. (2004a,b) established iAsIII as a transplacental carcinogen in C3H mice. These mouse strains are classical mouse models of cancer susceptibility, exhibiting high risks for both spontaneous and chemically induced liver cancer (Smith et al., 1973).
There is convincing evidence that arsenic is an important environmental carcinogen and that it is methylated during its metabolism (Cui et al., 2004a; Vahter, 2002). Hence, it has been proposed that arsenic metabolism may deplete intracellular methyl group stores and thereby lead to changes in DNA methylation that may be involved in carcinogenesis (Chen et al., 2004; Mass and Wang, 1997; Zhao et al., 1997). In the past few years, the important role of DNA methylation in carcinogenesis has been explored (Jones, 2003; Lund and van Lohuizen, 2002). This epigenetic modification occurs often in the promoter region of tumor suppressor gene and is associated with transcriptional silencing of the genes (Esteller, 2004; Jain, 2003).
The p16INK4a and RASSF1A are tumor suppressor genes frequently inactivated by de novo promoter hypermethylation in many cancer types including lung cancer (Kim et al., 2001; Wang et al., 2004). The p16INK4a gene was the first tumor suppressor gene found inactivated in lung cancer predominantly through aberrant hypermethylation of its CpG island. The p16INK4a gene is inactivated by methylation at prevalence up to 60–70% in lung adenocarcinomas. Lung tumors induced in F344/N rats after exposure to cigarette smoke by inhalation displayed de novo methylation of p16INK4a (Swafford et al., 1997, 2002). Methylation of the p16INK4a promoter region was also induced in 94% of adenocarcinomas of rats treated with the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (Belinsky et al., 1998). The RASSF1A gene, at 3p21.3, has been suggested to be a major target tumor suppressor on the basis of its frequent epigenetic silencing and loss of heterozygosity in lung cancers (Burbee et al., 2001).
Strain A/J mice have a relatively high spontaneous adenoma/adenocarcinoma incidence and, following exposure to a carcinogen, readily develop additional lung tumors that show multiple similarities in genetic alterations and signal transduction pathways to human lung adenocarcinoma (Bogen and Witschi, 2002; Wang et al., 2005). In the present study, we planned to establish iAsV carcinogenicity in spontaneous lung carcinogenesis in A/J mice. We also investigated the effects of prolonged exposure to iAsV on epigenetics such as DNA methylation status in the promoter region of p16INK4a and RASSF1A genes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Specific pathogen-free, 4-week-old male A/J mice (n = 120, CLEA Japan Inc., Tokyo, Japan) were acclimated to laboratory conditions in a temperature-controlled room at 24 ± 2°C under a 12 h (light)/12 h (dark) illumination cycle for 1 week prior to the start of the study. The mice were randomly divided into four groups of 30 animals each. Food and distilled water containing 0, 1, 10, and, 100 ppm of sodium arsenate (iAsV, Na2HAsO4·7H2O; Sigma, St. Louis, MO) were provided ad libitum for the experimental period. The animals were observed daily, and the nutritional condition and water intake were checked weekly throughout the experiment. All experiments were conducted in accordance with the institutional guidelines of the National Institute for Environmental Studies, Tsukuba, Japan.
Tissue collection.
After exposure to arsenic for 18 months, the animals were anesthetized ip with 40 mg/kg pentobarbital, and then the lungs were removed and weighed. Lung tissue samples were cut into two or three parts for pathological examination, arsenic analysis, and RNA/DNA extraction. Other organs such as liver, kidney, and skin were also removed and subjected to pathological examination.
Arsenic concentration in mice.
A portion of the lung (calculated to weigh about 20 mg) was digested with a mixture of HClO4-HNO3 solution (ratio 1:3 vol/vol) for 2 days at 130°C (Cui et al., 2004b). After removal of HNO3 by evaporation, the digested samples were diluted with deionized water and analyzed for arsenic by inductively coupled plasma–atomic emission spectroscopy (ICAP-61E-Trace, Thermo Jarrell-Ash, MA).
Pathology.
A portion of lung, liver, kidney, and skin was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin (HE) for histological examination. Pulmonary lesions were classified according to the criteria of classification of pulmonary lesions of mouse (Nikitin et al., 2004). All lung lesions in mice are delivered from type II pneumocytes (not Clara cells). Lung adenomas and tumors appeared to be solid/alveolar growth patterns, and no papillary adenomas/tumors were found. Briefly, hyperplastic lesions were microscopic and involved a minimum of 5–10 alveoli in a focus lined by hyperplastic type II epithelial cells. Adenomas were characterized by a monomorphic growth pattern and were generally composed of well-differentiated cells. Adenocarcinomas were composed of cells with various degrees of differentiation and were characterized by complete loss of normal architecture. Pathological assessments were conducted in a blind fashion.
Sodium bisulfite modification and methylation-specific polymerase chain reaction.
Genomic DNAs from lungs of animals were extracted using a DNeasy Tissue kit (Qiagen, Valencia, CA) according to the protocol. Bisulfite modification of DNA (1 µg) was carried out with the CpGenome DNA Modification kit (Intergen, Purchase, NY) according to the manufacturer's instructions. In this reaction, all unmethylated cytosines are converted to uracil, but methylated cytosines are resistant to this modification. Modified DNA samples were precipitated with ethanol and resuspended in a TE buffer (10mM Tris and 1mM EDTA [pH 7.5]) and used immediately or stored at –80°C until use.
The p16INK4a and RASSF1A promoter methylation status were determined by methylation-specific polymerase chain reaction (PCR). Methylation-specific PCR for p16INK4a and RASSF1A were conducted using primer sets for the methylated and unmethylated sequence (Table 1) and conditions described previously (Govindarajan et al., 2002; Tommasi et al., 2005).
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Reverse transcriptase–polymerase chain reaction.
Total RNA was extracted from the lung tissues of both control and iAsV-exposed mice using TRIZOL (Gibco BRL, Gaithersburg, MD). RNA purity and concentrations were determined by measuring A260/A280 absorption. cDNA was synthesized from 1 µg of RNA with a Thermoscript reverse transcriptase–polymerase chain reaction (RT-PCR) System (Gibco BRL, Gaithersburg, MD), and gene expression levels were measured with a semiquantitative RT-PCR using corresponding primers for GADPH, p16INK4a, and RASSF1A (Table 2) (Roca et al., 2003
; Tommasi et al., 2005). In brief, specific GADPH primers were used for the internal control to normalize the sample amounts. PCR was performed in the thermal cycler (GeneAmp PCR System 9700, ABI, Foster, CA) for 29–40 cycles consisting of denaturation at 94°C for 30 s, annealing at 58°C GADPH for 45 s or 63°C (p16INK4a and RASSF1A primer sets) for 30 s and extension at 72°C for 1 min followed by a final 5-min extension at 72°C. The reaction products were loaded onto 1.8% agarose gels containing ethidium bromide and visualized under UV illumination.
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Immunohistochemistry.
For immunohistochemistry, sections cut from formalin-fixed, paraffin-embedded tissue blocks were deparaffinized and rehydrated through a graded series of ethanol. The sections were then processed in a 10mM citrate buffer (pH 6.0) and heated in an autoclave for 10 min for antigen retrieval. The slides were preincubated with normal horse serum (1:50 dilution; Vector Laboratories, Burlingame, CA) to diminish nonspecific binding of the secondary antibody. The slides were then incubated overnight at 4°C with mouse monoclonal anti-p16INK4a (mouse monoclonal, BD Pharmingen, San Diego, CA) and anti-RASSF1A (goat polyclonal, Santa Cruz, Santa Cruz Biotechnology, CA) at a dilution of 1:500. Slides were then rinsed and incubated with universal secondary antibody containing anti-mouse/anti-goat IgG (Vectastain ABC Elite kit; Vector Laboratories) for 30 min, developed with diaminobenzadine (Vectastain ABC Elite kit; Vector Laboratories) for 20 min, and counterstained with hematoxylin for 1 min. Nuclear reactivity for p16INK4a and RASSF1A proteins was considered as positive or negative as described previously (Cui et al., 2004c
). Briefly, sections were examined for evidence of nuclear staining above any cytoplasmic background. If there was reactivity in the nontumor tissue, the sample was considered to be uninterpretable. For p16INK4a: –, 0%; +, 1–50%; ++, > 50%; for RASSF1A: –, 0%; +, 1–30%; ++, > 30%; % indicates the percentage of the nuclear immunostained lung cells with each individual protein.
Statistical study.
Student's t-test was used to determine the differences in the number and size of lung tumors per mouse between iAsV-exposed and control mice. The lung tumor grades were ranked, and the data were evaluated by Wilcoxon analysis using Statview version 5.0 software. Numerical data are expressed as the mean ± SD and were analyzed by ANOVA and multivariate analysis. Significance was set at the p < 0.05 level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A/J Mice Survival and Arsenic Concentration in Lung and Liver Tissues
Some of the mice exposed to 0 (control), 1, and 10 ppm of iAsV began to die after 10 months, and the survival rate of the 0, 1, 10, and 100 ppm iAsV–exposed mice at the end of the experiment was 63% (19/30), 47% (14/30), 53% (16/30), and 100% (30/30), respectively (Fig. 1). Although the reason for the mouse deaths is unclear, skin lesions accompanied by inflammation were significantly associated with mice survival. Interestingly, no skin lesions were found in the high dose of iAsV-exposed mice (100 ppm). Body weight was reduced in the 1 and 100 ppm iAsV–exposed mice compared to that of the control, whereas the lung weight and lung/body weight ratio increased in the 1 and 10 ppm iAsV–exposed mice (Fig. 2). Total arsenic concentrations in the lung and liver tissues were increased dose dependently in iAsV-exposed mice (Fig. 3). Although no dose-related effects on body weight or lung weight were evident, iAsV was slightly toxic at all dose levels tested (disrupted hepatic cords and dilated sinusoidal were observed in iAsV-exposed mice).
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iAsV-Induced Lung Tumor and Pathological Analysis
The iAsV exposure to A/J mice significantly increased additional lung tumor numbers and tumor size. Four hyperplasias were observed in the control, whereas two hyperplasias and four adenomas were found in 100 ppm iAsV–exposed mice. No hyperplasias and adenomas were found in the 1 and 10 ppm iAsV–exposed mice. An increase in the number of lung tumors was apparent in all groups receiving iAsV, with the greatest severity in the group receiving the 100 ppm of iAsV (Table 3). Total numbers of tumors in iAsV-exposed mice were twofold higher than in the control (Fig. 4a). The percentage of tumors with size more than 4 mm was increased in a dose-related manner in the iAsV-exposed mice compared to the control mice (Fig. 4b). Histopathological examination showed that the rate of poorly differentiated lung adenocarcinoma was much higher in iAsV-exposed mice than in the control (Table 4).
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Pathological analysis of other tissues revealed no obvious histological abnormalities in a range of organs except for one liver cancer observed in the control and 100 ppm iAsV–exposed mice and one skin cancer found in the 1 and 10 ppm iAsV–exposed mice.
CpG Methylation Status of p16INK4a and RASSF1A
The current study used methylation-specific PCR to examine the methylation status of the p16INK4a and RASSF1A genes in lung tissues with hyperplasias, adenomas, and adenocarcinomas from the control and iAsV-exposed mice. Methylation of promoter regions of the p16INK4a and RASSF1A was also detected in nontumor lung tissues (including hyperplasia and adenoma) both from the control and 100 ppm iAsV–exposed mice, though no methylation was detected in the 1 and 10 ppm iAsV–exposed mice. However, methylation rates increased in a dose-dependent tendency in lung adenocarcinomas from iAsV-exposed mice compared to the control (Table 5). Methylation of the p16INK4a gene was detected in 1 of 9 adenocarcinomas in the control, compared to 3 of 10, 4 of 11, and 8 of 19 adenocarcinomas in the 1, 10, and 100 ppm iAsV–exposed mice, respectively. Methylation of RASSF1A was detected in 3 of 9 adenocarcinomas in the control, compared to 7 of 10, 9 of 11, and 17 of 19 adenocarcinomas in the 1, 10, and 100 ppm iAsV–exposed mice, respectively (Table 5). Figure 5 shows a representative methylation status of p16INK4a and RASSF1A in lung tissues from control and iAsV-exposed mice. As shown in Figure 5, both unmethylated and methylated p16INK4a alleles were detected by methylation-specific PCR in nontumor lung tissues from the control mice. Relatively, high rate of hypermethylation of the genes was observed in lung tumor tissues from iAsV-exposed mice.
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mRNA Levels of p16INK4a and RASSF1A
The mRNA levels of p16INK4a and RASSF1A were examined by semiquantitative RT-PCR, and a weak or loss of p16INK4a and RASSF1A expression was found in lung tumors of control and iAsV-exposed mice, whereas a constant expression of p16INK4a and RASSF1A in nontumor lungs was found in both control and iAsV-exposed mice. Lower or loss of p16INK4a and RASSF1A expression was closely correlated with the methylation of these two genes. Figure 6 shows representative photographs of the agarose gels for each RT-PCR product stained with ethidium bromide. There was no correlation between p16INK4a and RASSF1A expressions, indicating that the two genes were regulated independently.
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Morphology, Histology, and Immunohistochemistry
Figure 7a shows a representative lung tumor and its HE staining in the control, 1, 10, and 100 ppm iAsV–exposed A/J mice. As seen in the Figure 7a, lung tumors induced by iAsV exposure were much bigger than those in the control. All histological grades presented are poorly differentiated adenocarcinoma. The relationship between arsenic exposure and expression of the p16INK4a and RASSF1A was assessed through immunohistochemical analysis. Figures 7b and 7c show a representative p16INK4a and RASSF1A immunohistochemical staining in nontumor tissues and tumors. Only nuclear staining was considered as positive. Lower or loss of p16INK4a and RASSF1A expression in lung tumors was found in iAsV-exposed mice, compared to a modest expression of p16INK4a and RASSF1A in nontumor lungs in both control and iAsV-exposed mice (Table 6). In a nontumor that was unmethylated at the p16INK4a and RASSF1A locus, modest to strong positive expression for p16INK4a and RASSF1A proteins were observed. In contrast, weak or a complete lack of p16INK4a and RASSF1A staining was observed in adenocarcinoma that was methylated (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We found that exposure to iAsV caused arsenic accumulation in the lung and developed additional lung tumors in A/J mice. Histopathological examination showed that an increased rate of poorly differentiated adenocarcinoma was observed in iAsV-exposed mice. High doses of arsenic seem to be more carcinogenic to the mice because the tumor size in 100 ppm iAsV–exposed mice is more larger than in the 1 and 10 ppm iAsV–exposed mice. The number of tumor size more than 4 mm significantly increased in a dose-related manner in iAsV-exposed groups. Although different genetic background between mouse and human exists, lung tumors occurring in A/J mice are highly relevant for the study of human lung adenocarcinomas because they originate from the same cells, the type II alveolar epithelial cells and Clara cells (Witschi et al., 2002
). They also share many of the molecular changes found in cell cycle control genes, have obvious similarities in certain enzyme activities and other biochemical processes, and share other genetic and epigenetic features (Witschi, 2005).
The iAs undergoes a distinct biotransformation pathway in the body mediated through a methylation process. Arsenic methylation requires S-adenosyl-methionine (SAM) as a methyl group donor and arsenic methyltransferase (Hayakawa et al., 2005). SAM is an essential cofactor for a variety of methyltransferases in the cell including DNA methyltransferases, which are responsible for the methylation of DNA. Exposure of human lung tumor cells to iAs produced significant dose-responsive hypermethylation of the promoter of p53 gene (Mass and Wang, 1997). Recently, it has also been shown that a significant hypermethylation of p53 and p16INK4a genes was observed in patients with arsenicosis or arsenic-induced skin cancer (Chanda et al., 2006). In the present study, we assessed methylation status of promoter regions of p16INK4a and RASSF1A in lung tissues from iAs-exposed mice. Hypermethylation of the p16INK4a and RASSF1A CpG islands was detected in nontumor lung tissues (including hyperplasia and adenoma) from the control and 100 ppm iAsV–exposed mice, and the hypermethylation rates of the genes appeared to be higher in a dose-related tendency in lung adenocarcinomas from iAsV-exposed mice compared to that from the control. These data implicate that chronic oral exposure to iAsV epigenetically interfered with DNA methylation patterns and that it may be attributable to the development of lung tumorigenesis in A/J mice. This finding is in agreement with previous reports that high frequent hypermethylation of the p16INK4a and RASSF1A genes was detected in lung cancer both in human and animal models (Belinsky 1997, 2005; Burbee et al., 2001; Kim et al., 2001; Wang et al., 2004).
It has been reported that hypermethylation of the p16INK4a gene is frequently observed in non-small cell lung cancer, and 50% of losses of p16INK4a expression in non-small cell lung cancer can be caused by hypermethylation of the promoter region in the p16INK4a gene (Gazzeri et al., 1998; Merlo et al., 1995). Many observations suggest that RASSF1A inactivation is closely related to Ras activation in human cancers and thus contributes to malignant transformation by inhibiting Ras-mediated apoptosis (Dammann et al., 2000). RASSF1A blocks G1-S cell cycle progression by inhibiting the accumulation of cyclin D1 at the level of translational control. The inactivation of RASSF1A by CpG island hypermethylation induced cyclin D1 accumulation and direct cell cycle progression positively (Shivakumar et al., 2002). Immunohistochemical study showed that a weak or loss of p16INK4a and RASSF1A expression was found in lung tumors of control and iAsV-exposed mice, whereas a constant modest to strong expression of p16INK4a and RASSF1A in nontumor lungs was found in both control and iAsV-exposed mice. Reduced expressions of the genes in the lung tumors both from control and iAsV-exposed mice were observed, and these lower or loss of p16INK4a and RASSF1A expression was associated with hypermethylation of these two genes. This finding suggests that iAsV exposure modulates gene expression even at protein levels through alteration of DNA methylation patterns of the genes. These results in line with previous studies report that altered expression of p16INK4a and RASSF1A may be an important step in early neoplastic transformation of the lung because hypermethylation at these regulatory regions may contribute to lung carcinogenesis by facilitating decreased expression and cell cycle dysregulation (Belinsky et al., 1998; Burbee et al., 2001). All together, our data indicate that altered DNA hypermethylation of the p16INK4a and RASSF1A CpG islands induced by arsenic plays an pivotal role in the development of lung tumorigenesis.
Transcriptional silencing by CpG island hypermethylation now rivals genetic changes that affect coding sequence as a critical trigger for neoplastic development and progression (Esteller, 2004; Lund and van Lohuizen, 2002). Genes responsible for all aspects of normal cellular function are targeted for inactivation by methylation. Besides promoter hypermethylation of the tumor suppressor genes, involvement of genomic DNA hypomethylation in tumorigenesis has also been demonstrated. It has been reported that chronic exposure of iAs induces global DNA hypomethylation and aberrant gene expression in a rat liver epithelial cell line in vitro (Zhao et al., 1997) and in C57BL/6J and 129/SvJ mice liver in vivo (Chen et al., 2004; Okoji et al., 2002). Taken together, both DNA hypo- and hypermethylation are linked to arsenic-induced malignant transformation and carcinogenesis. However, many other possible mechanisms of lung tumorigenesis have been postulated (Belinsky et al., 2003; Lund and van Lohuizen, 2002). Further study of this issue should be carried out.
In summary, chronic exposure to iAsV leads to arsenic accumulation, alters expression of p16INK4a and RASSF1A through influencing methylation patterns of the genes, and induced additional lung tumors in A/J mice.
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ACKNOWLEDGMENTS |
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This study was supported in part by Grants-in-Aid for Scientific Research (Young Scientists B, No.14780433) from the Japan Society for the Promotion of Science (to X.C.).
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