Sodium arsenite administration via drinking water increases genome-wide and Ha-ras DNA hypomethylation in methyl-deficient C57BL/6J mice
R.S. Okoji1,
R.C. Yu1,
R.R. Maronpot2 and
J.R. Froines1,3
1 Center for Occupational and Environmental Health, UCLA School of Public Health, 650 Charles E. Young Drive South, Los Angeles, CA 90095 and
2 National Institute of Environmental Health Sciences, Laboratory of Experimental Pathology, 111 T. W. Alexander Drive, MD B3-06, Research Triangle Park, NC 27709, USA
 |
Abstract
|
---|
Arsenic is an established human carcinogen. Deficiencies in available animal models have inhibited a detailed analysis of the mechanism of arsenic induced cancer. This study sought to determine the role of a methyl-deficient diet in combination with sodium arsenite on the genomic methylation status and Ha-ras methylation status of C57BL/6J male mice hepatic DNA. Mice were administered arsenic as sodium arsenite via drinking water at 0, 2.6, 4.3, 9.5 or 14.6 mg sodium arsenite/kg/day. Administration occurred 7 days a week for 130 days. Dose-related effects on the liver were evident in mice administered arsenic and methyl-deficient diets. Most prominent were observations of steatosis and microgranulomas. Sodium arsenite increased genomic hypomethylation in a dose dependent manner and methyl-deficiency and sodium arsenite reduced the frequency of methylation at several cytosine sites within the promoter region of the oncogenic gene, Ha-ras. Methylation changes were prominent in a 500 bp non-CpG island-like region of the Ha-ras promoter and less prominent in a 525 bp CpG island-like region. DNA methylation plays an important role in the physiological expression of many genes including Ha-ras. Significantly reduced methylation at a key regulatory region of Ha-ras in the mouse liver may have relevance to understanding arsenic-induced perturbations in the methylation patterns of cellular growth genes involved in the formation of tumors. These findings highlight the effect of sodium arsenite on inherent methylation processes within the hepatic cell.
Abbreviations: MDD, methyl-deficient diet; MSD, methyl-sufficient diet; SAM, S-adenosyl-methionine.
 |
Introduction
|
---|
Arsenic is classified as a human carcinogen by the International Agency for Research on Cancer (IARC), the United States Environmental Protection Agency (USEPA), Occupational Safety and Health Administration (OSHA), and the National Toxicology Program (NTP). In exposed populations, inorganic arsenic is associated with tumors of the lung, liver, bladder, kidney and skin (16). However, the mechanism of arsenic carcinogenesis has remained an enigma. Although identified as a human carcinogen, the carcinogenicity of arsenic in animals has never been convincingly demonstrated. Earlier chronic animal bioassays have either failed to induce tumors in laboratory animals or were flawed or incomplete for establishing carcinogenicity (79).
Inorganic arsenic undergoes a distinct biotransformation pathway in the body mediated through an oxidative methylation process. Both mono-and dimethylated metabolites are formed and excreted after ingestion of inorganic forms. Arsenic methylation requires S-adenosyl-methionine (SAM) as a methyl group donor and the recently characterized arsenic methyltransferase (10). Humans are efficient methylators of arsenic with methylation occurring primarily in the liver, a suspected target organ for arsenic induced cancer (11). SAM is an essential cofactor for a variety of methyltransferases in the cell including DNA methyltransferases, which are responsible for the methylation of DNA. DNA methylation is involved in the control of gene expression and may have implications in carcinogenesis.
Several observations implicate DNA methylation in the carcinogenic process. First, human tumors normally exhibit decreased overall genome methylation (1214) with specific regions of either hypo-or hypermethylation (1517). Second, hypomethylation has been observed to be a relatively early event in colorectal cancer (18) and the extent of hypomethylation appears to increase progressively along with the stage of malignancy (19,20).
DNA methylation is part of a complex regulatory system for the expression of genes including those involved in cellular growth and differentiation (21). DNA hypomethylation is associated with DNA conformational changes and increased gene expression (22,23). Changes in the expression of cell-cycle control genes can directly result in cell transformation (2426). Evidence of this is seen with the administration of the nongenotoxic carcinogen, phenobarbitol, to B6C3F1 mice. Phenobarbital significantly increases the incidence of tumors in the liver. Examination of Ha-ras in tumor tissue indicates reduced methylation at CpG sites (15,27) and increased expression of the gene (28).
DNA methylation can be altered by dietary manipulation of methyl donors. Animals exposed solely to methyl-deficient diets display perturbations in hepatic methyltransferase activity (29). Measurements of 5-methylcytosine in the DNA hydrolysates of methyl-deficient animals confirm a reduction in DNA methylation (30,31). DNA hypomethylation is induced in both B6C3F1 and C57BL/6J mice fed a methyl-deficient diet for as little as 1 week. B6C3F1 mice display a 21% decrease in liver DNA methylation status and C57BL/6J mice exhibit a 9% reduction (32).
It is plausible that a reduction in DNA methylation as a consequence of dietary induced methyl-deficiency may result in increased expression of cell-cycle control genes and tumor development. Male Fisher rats on methyl-deficient diets present with reduced DNA methylation and increased levels of the mRNA of c-myc, c-fos, and c-Ha-ras, genes known to be involved in the transformation process of many human tumors (29,3335). Hsieh and coworkers (36) have shown a similar association with c-myc and c-Ha-ras in B6C3F1 mice. Lung and liver tumors have been induced in rats chronically exposed to methyl-deficient diets (3743). Thirteen-month old Fisher 344 male rats on methyl-deficient diets for 12 months present with 100% incidence of preneoplastic hepatocyte nodules and a 51% incidence of hepatocellular carcinoma (43). Mice fed choline-deficient diets for a period of 172 days also develop tumors in the liver (44).
Together, these evidences suggest that arsenic metabolism may be relevant in the depletion of methyl stores available for DNA methylation with subsequent implications for carcinogenesis related to hypomethylation. To test this hypothesis we used methyl-deficient C57BL/6J mice administered sodium arsenite via drinking water to investigate the effects of arsenic on genome-wide and Ha-ras specific hypomethylation.
 |
Materials and methods
|
---|
Experimental design
Ninety young-adult male C57BL/6J mice ~8 weeks old were obtained from Jackson Laboratories and housed three to a cage in the UCLA School of Public Health vivarium according to the NIH `Guide for the Care and Use of Laboratory Animals'. Cages were polycarbonate, shoebox style with wire lid and filter covers to provide an isolated environment by minimizing variation in temperature, humidity and drafts. Mice were maintained on a lab chow diet during a 1-week quarantine period in which clinical signs of illness were closely monitored. Clinical examinations included a twice daily check for appearance, morbidity and mortality, posture or behavior. Periodic tests for neurological and ophthalmological response were also conducted by the campus veterinarian. Only clinically healthy mice were used in the study. After the quarantine period, mice were transferred to a negatively adjusted air pressure room maintained on a 12 h light/12 h dark schedule at a constant temperature of 23.3°C ± 1.1 and a relative humidity of 40 ± 5%. Mice were then placed randomly into one of five treatment groups and pretreated with a methyl-deficient diet (MDD) for a period of 21 days. No attempt was made to normalize different groups by body weight.
The study incorporated two control groups: one group was maintained on a methyl-sufficient diet (MSD) for the length of the study while a second group was maintained on a methyl-deficient diet (MDD). The four remaining groups were maintained on methyl-deficient diets and administered arsenic as sodium arsenite (Sigma Chemical Company, St Louis, MO) in drinking water at concentrations ranging from 2.6 to 14.6 mg/kg/day. Treatment groups are listed below:
- Group 1: ad libitum on a MSD; no arsenic.
- Group 2: ad libitum on a MDD; no arsenic.
- Group 3: ad libitum on a MDD; 2.6 mg/kg/day sodium arsenite.
- Group 4: ad libitum on a MDD; 4.3 mg/kg/day sodium arsenite.
- Group 5: ad libitum on a MDD; 9.5 mg/kg/day sodium arsenite.
- Group 6: ad libitum on a MDD; 14.6 mg/kg/day sodium arsenite.
After 130 days the animals were killed, and their livers removed and stored at 70°C for analysis.
Diets
All diet ingredients were synthesized by ICN Biochemicals (Cleveland, OH). AIN 76 vitamin and mineral mixes were supplied by Sigma Chemical (St Louis, MO). The MDD diet contained low methionine (12% vitamin free casein), choline (10% soy oil) and folate (folate-free vitamin mix) (45). The MSD diet incorporated a methyl-deficient diet supplemented (MSD) with 0.4% L-methionine, 0.4% choline bitartrate and 2 mg/kg folic acid. Detailed compositions of MDD and MSD diets are shown in Table I
.
Histopathological examination
At necropsy, tissues were weighed and sections of livers, lungs, bladders, kidneys and skin were snapped frozen in liquid nitrogen for nucleic acid analysis. Additional tissue samples were fixed in neutral buffered formalin, stored in 70% alcohol and shipped to the National Institute of Environmental Health Sciences (NIEHS) where representative portions of the liver were trimmed, routinely processed, and sections stained with hematoxylin and eosin for histopathological examination.
Determination of global methylation
The use of Sss1 methylase provides an accurate tool for assessing changes in the extent of DNA methylation as a result of dietary manipulation and arsenic administration because it methylates both hemimethylated and unmethylated cytosines in genomic DNA with equal efficiency. Incorporation of radiolabelled S-adenosyl-methionine is directly proportional to the degree of DNA methylation and DNA concentration. The extent of SAM incorporation is measured via scintillation counting and reported in counts per minute (c.p.m.), and evaluation of methylation status of different samples can be compared based on relative c.p.m.
Global methylation assays were conducted by the method of Balaghi and Wagner (30). Genomic DNA was isolated by the method of Couse et al. (46) with slight modifications. 50 mg of liver tissue was homogenized in 0.6 ml of lysis buffer containing 100 mM NaCl, 10 mM Tris/HCl (pH 8.0), 25 mM EDTA (pH 8.0), 0.5% SDS and 0.1 mg/ml proteinase K. The digest was vortexed for 10 s and then put in a heating block set to 55°C overnight. Following the digestion, digests were transferred to a serum separation tube and an equal volume of phenol/chloroform (1:1) was added. Mixtures were inverted on a rocking platform for 10 min and then centrifuged. DNA was ethanol precipitated and resuspended in 200 ml of TE buffer (pH 8.0). Final preparations had an absorbance ratio 280/260 between 1.8 and 2.0. Isolated DNA was then incubated with bacterial CpG methylase (Sss1 methylase) (New England Biolabs) and radiolabelled S-adenosyl-methionine (Amersham Pharmacia Biotech), which specifically methylates cytosine residues at the 5' position of CG sequences. Incorporated radiolabel, representing degree of methylation, was quantified by scintillation counting.
The experimental conditions were modified from the original protocol to maximize the methylation reaction. The reaction mixture contained: DNA, 0.5 mg; S-adenosyl-L-[methyl3H]methionine, 1 mCi; SssI methylase, 2 units; NE Buffer 2 containing 50 mM NaCl, 10 mM Tris/HCl, 10 mM MgCl2, and 1 mM DTT. The final volume was brought up to 30 ml. The mixture was prepared in duplicate and incubated for 1 h at 30°C.
The reactions were processed four times and terminated by filtering 15 ml on Whatman DE 81 ion exchange filter paper fitted on a vacuum apparatus, washed 3 times with 0.5 M sodium phosphate buffer (pH 7.0), 2 times with 2 ml 70% alcohol and 2 ml of absolute ethanol. The filters were dried and placed into a scintillation vial containing 5 ml of Scintisafe aqueous counting scintillant (Fisher Scientific). Blank values were obtained from incubations without CpG methylase.
Determination of Ha-ras-specific methylation
Methylation status of the Ha-ras gene was examined in the livers of all sacrificed animals by methylation-sensitive restriction analysis and PCR of the purified tissue DNA as detailed in Counts et al. (47). One microgram of genomic DNA was digested to completion with 5 units/µg of methylation sensitive restriction enzyme in two 2.5 unit aliquots at 3 h intervals. PCR primers were designed to flank the site of interest. If a site was methylated, a PCR product for a particular primer pair was evident. If a site was unmethylated, amplification of the region did not occur. Restriction enzymes included: HaeII, SmaI, HaeIII AluI, StuI, EcoRII, AvaII, MspI, HpaII, HhaI and XhoI. Their specific sites of recognition and non-recognition are given in Table II
. Restriction endonuclease digested DNA was then subject to enzyme and salt removal using Micropure-EZ with Microcon concentrator (Millipore®). The purified DNA was resuspended in 10 µl of TE buffer (pH 8.0) and subject to PCR. Each 50 microliter PCR reaction contained 0:10 pmol (pm) each primer, 0.05 µg of DNA, 0.5 units of Taq polymerase, 1.5 mM MgCl2 and 25 pmol dNTPs. PCR cycling conditions were as follows: 5 min at 80°C, 2 min at 94°C, 24 cycles (96°C for 60 s, 65°C for 75 s, 72°C for 120 s), 72° for 5 min. Primer pairs were constructed to encompass both a non-CpG island (Segment Ras 474/976: Sense 5'-GCGACCGGGG-TGAGCGTGCAA-3', Anti-sense 5'-AGAGCCTCCACCCTGCAGCCT-3') and a CpG island (Segment 962/1487: Sense 5'-CAGGGTGG-AGGCTCTGTAGT-3', Anti-sense 5'-GAGAGGAGCAAGGAAGCACC-3'). The regions amplified by primer sets 474/976 and 962/1487 correspond to base pairs in the 2331 bp region of the 5' flanking region of Ha-ras identified by Counts et al. (47) and previously by Brown et al., (48) and Neades et al. (49). PCR cycling conditions were maintained at 25 cycles to maximize identification of heterogeneous sites of methylation (i.e., some cells in the sample were methylated at the site of interest, while other cells were unmethylated at the site of interest). Carrying out the PCR reaction on a partially methylated site the conventional 40 cycles would lead to an amplification of product indistinguishable from sites fully methylated. In this way, heterogeneity of methylation was evident by the variable intensities of PCR product. Control lanes using known methylated, unmethylated and partially methylated sites (47) were run alongside unknown sites of methylation to compare the degree of methylation at that particular site. Methylation was classified as either fully methylated, unmethylated, partially methylated (predominantly methylated, greater than half the intensity of methylated control) and partially methylated (predominantly unmethylated, less than half the intensity of methylated control). Following amplification, PCR reactions were electrophoresed on a 3% agarose gel, 1x TBE and visualized under ethidium bromide fluorescence.
View this table:
[in this window]
[in a new window]
|
Table II. Methylation sensitive restriction enzymes and their sites of recognition and non-recognition used in the assessment of the methylation status of Ha-ras in methyl-deficient C57BL/6 mice administered sodium arsenite
|
|
Statistical methods
The prevalence of histopathological changes in the livers of methyl-sufficient mice and methyl-deficient mice treated with arsenic are presented in Tables III and IV
as the numbers of animals observed with varying degrees of fatty change and microgranulomas in livers and the total number of animals examined (pathological changes were graded from 1 to 4: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked). Prevalence rates of the histopathological changes were also calculated. Changes in 5-methyl-cytosine content in segments amplified by primer set 474/976 and 962/1487 of Ha-ras were graded in Tables V and VI
, respectively, as follows: 1 for totally unmethylated; 2 for predominantly unmethylated (the intensity of segment signals was less than half of that of methylated control); 3 for predominantly methylated (the intensity of segment signals was greater than half of that of methylated control); and 4 for fully methylated. Means and standard error (SE) of these values for all animals for each group were calculated. MannWhitney tests (50) were used to compare methylation status between the methyl-deficient group (MDD) and the methyl-sufficient group (MSD) and between methyl-deficient group plus various arsenic concentrations (MDD + arsenic conc.) and methyl-deficient group only (MDD). The former was to test the effect due to methyl-deficient diet only, while the latter was to delineate arsenic effects. SAS PROC NPAR1WAY with Wilcoxon option (SAS Institute) was used to implement these tests. Two-sided P > 0.05 implies that the effects due to the methyl-deficient diet only or due to arsenic exposures was not significant. P
0.05 suggests there is an effect resulting from the methyl-deficient diet or arsenic exposure (see Tables V and VI
). Two-sided Student's t-tests were conducted to compare the effect of global methylation due to methyl-deficient diet and increasing doses of arsenic.
View this table:
[in this window]
[in a new window]
|
Table III. Number of animals and prevalence (%) of fatty changes in livers of mice treated with methyl-sufficient diet and methyl-deficient diets plus arsenic
|
|
View this table:
[in this window]
[in a new window]
|
Table IV. Number of animals and prevalence (%) of microgranulomas in livers of mice treated with methyl-sufficient diet and methyl-deficient diets plus arsenic
|
|
View this table:
[in this window]
[in a new window]
|
Table V. Changea in 5-methyl-cytosine content in segment 474/976 of Ha-ras in methyl-deficient C57BL/6 mice administered sodium arsenite
|
|
View this table:
[in this window]
[in a new window]
|
Table VI. Changea in 5-methyl-cytosine content in segment 962/1487 of Ha-ras in methyl-deficient C57BL/6 mice administered sodium arsenite
|
|
 |
Results
|
---|
Although no treatment related effects on bodyweight or liver weight were evident, sodium arsenite was moderately toxic at all dose levels tested. Steatosis of the liver, characterized by singular clear vacuoles consistent with macrovesicular fatty droplets was observed in mice administered methyl-deficient diets and sodium arsenite (Figure 1
, Table III
). An increase in the number of microfoci of hepatic inflammation (microgranulomas) was also apparent in all groups receiving a methyl-deficient diet with the greatest severity in the group receiving arsenic at 4.3 mg/kg/day (Table IV
). Severe hepatic parenchymal damage was seen in two arsenic-treated mice and is believed to be a consequence of repeated toxic insult and loss of hepatocytes (data not shown).

View larger version (158K):
[in this window]
[in a new window]
|
Fig. 1. (A) Liver from a control mouse (methyl-sufficient diet without arsenic) showing absence of fat vacuoles. (B) Mild hepatic steatosis in a mouse on a methyl-deficient diet plus 4.3 mg/kg/day of arsenic in the drinking water. Note the clear vacuoles representing fat deposition. (C) Moderate steatosis in the liver of a mouse on a methyl-deficient diet plus 9.5 mg/kg/day of arsenic in the drinking water. There is an increase in the number of fat vacuoles, most of which are concentrated around the central vein. (D) Marked hepatic steatosis in a mouse on a methyl-deficient diet plus 14.6 mg/kg/day of arsenic in the drinking water. Numerous fat vacuoles are present throughout the hepatic lobule.
|
|
Genomic DNA from livers of animals maintained on methyl-sufficient diets had a mean value of 1728 ± 469 c.p.m. Liver DNA from mice maintained on methyl-deficient diets exhibited a mean value of 2297 ± 557 c.p.m. Sodium arsenite administration further reduced genomic DNA methylation and this effect was found to be dose-dependent. Mice dosed with 2.6, 4.3, 9.5, and 14.6 mg/kg/day of sodium arsenite exhibited mean values of 2944 ± 223, 3273 ± 308, 3603 ± 517, and 3154 ± 449 c.p.m. respectively. Statistically significant differences (P < 0.05) were found between both methyl-sufficient and deficient mice and between methyl-deficient mice and methyl-deficient mice treated with arsenic (Figure 2
).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2. SssI methylase was used to assess methylation status indirectly. Incorporation of radiolabelled S-adenosyl-methionine (c.p.m.) is proportional to the degree of DNA hypomethylation and DNA concentration. Means identified by a letter are statistically different from one another. P < 0.05.
|
|
Methylation sensitive PCR was employed to analyze two 500 bp regions of the 5' flanking region of the oncogene Ha-ras for methylation status. CpG islands are defined as segments of DNA that remain free of methylation and are approximately 500 to 2000 bp in length with a >50% GC content and a high ratio of observed to expected CpG sites (23). The region amplified by primer set 474/976 begins 815 bp upstream from the 5' transcriptional start site (48) and has a relatively low CpG observed/expected ratio. It is therefore not considered a CpG island. Segment 962/1487 contains the most 5' transcriptional start site and has a greater than expected CpG ratio characteristic of CpG islands (47).
Results in Table V
showed the region amplified by primer set 474/976 was heterogeneously methylated. Degree of methylation at a particular CpG site was determined by the intensity of the band amplified by the two primer sets. Eleven methylation sensitive enzymes were employed in the examination of region 474/976. Only three sites were classified as completely methylated (HaeII) or completely unmethylated (HaeIII and SmaI). The remaining sites were classified as partially methylated (primarily methylated or primarily unmethylated). Differences in methylation status between animals fed the methyl-sufficient and methyl-deficient diets were observed at three sites. Significant reductions in 5-methyl-cytosine were found in animals fed the methyl-deficient diet at both AvaII and XhoI sites, while a significant increase in methylation was found at the site cleaved by EcoRII. Sodium arsenite treatment resulted in reductions in five of the 11 restriction sites examined when compared to animals maintained on methyl-deficient diets and not administered arsenic (EcoRII, StuI, AluI, AvaII, XhoI). Increased methylation was found at one site recognized by HhaI. Figure 3
depicts the amplified products of a representative sample of an MDD control animal for region 474/976. Digestion of liver DNA with the methylation sensitive enzymes HaeIII, StuI and SmaI prevented amplification of the region in the subsequent PCR indicating the lack of methylation at those sites. Pre-PCR digestion with HaeII, EcoRII, AvaII and XhoI had no effect on amplification, which is consistent with those sites being fully methylated. Heterogeneous methylation is seen in lanes digested by HpaII, MspI, AluI and HhaI. Figure 4
shows the amplified products of an animal maintained on the methyl-deficient diet and administered 9.5 mg/kg/day sodium arsenite via drinking water. Digestion of liver DNA with StuI or AvaII prevented amplification of segment 474/976 by PCR. Incubations with EcoRII and XhoI resulted in PCR products of less intensity than undigested controls indicating a heterogeneously methylated (primarily unmethylated) sample of cells.

View larger version (87K):
[in this window]
[in a new window]
|
Fig. 3. Lane 1, positive control (site known to be methylated in untreated C57BL/6 mice; from Countset al., 1997). Lane 2, negative control (site known to be unmethylated in untreated C57BL/6 mice). Lane 3, partially methylated control (site known to be partially methylated in untreated C57BL/6 mice). Even numbered lanes represent the amplified products of primer set 474/976 without prior digestion. Pre-PCR digestion of liver DNA with methylation sensitive enzymes HaeIII (Lanes 17 and 19), StuI (Lanes 25 and 27) and SmaI (Lanes 45 and 47) prevented amplification of region 474/976. Pre-PCR digestion with HaeII (Lanes 13 and 15), EcoRII (Lanes 21 and 23), AvaII (Lanes 33 and 35) and XhoI (Lanes 37 and 39) had no effect on amplification, which is consistent with those sites being fully methylated. Heterogeneous methylation was seen in lanes digested by HpaII (Lanes 5 and 7), MspI (Lanes 9 and 11), AluI (Lanes 29 and 31) and HhaI (Lanes 41 and 43).
|
|

View larger version (79K):
[in this window]
[in a new window]
|
Fig. 4. Lane M, bp ladder (from bottom, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 bp). Lane 1, positive control (site known to be methylated in untreated mice; from Counts et al., 1997). Lane 2, negative control (site known to be unmethylated in untreated C57BL/6 mice). Lane 3, partially methylated control (site known to be partially methylated in untreated C57BL/6 mice). Even numbered lanes represent the amplified products of primer set 474/976 without prior digestion. Pre-PCR digestion of liver DNA with methylation sensitive enzymes HaeIII (Lanes 17 and 19), StuI (Lanes 25 and 27), AvaII (Lanes 33 and 35) and SmaI (Lanes 45 and 47) prevented amplification of region 474/976. Pre-PCR digestion with HaeII (Lanes 13 and 15) and HhaI (Lanes 41 and 43) had no effect on amplification, which is consistent with those sites being fully methylated. Heterogeneous methylation was seen in lanes digested by HpaII (Lanes 5 and 7), MspI (Lanes 9 and 11), EcoRII (Lanes 21 and 23), AluI (Lanes 29 and 31) and XhoI (Lanes 37 and 39).
|
|
Results show that the region amplified by primer set 962/1487 was predominantly devoid of methylation as six out of 10 examined sites were classified as unmethylated. A representative sample from the MDD and MDD + 9.5 mg/kg groups for the region amplified by primer set 962/1487 are given in Figures 5 and 6
, respectively. Four sites (MspI, HaeIII, XhoI and SmaI) were classified as partially methylated (primarily unmethylated). A significant reduction in the occurrence of 5-methyl cytosine (P < 0.05) was observed for the site recognized by SmaI between MSD and MDD treatment groups. This site was partially methylated (primarily unmethylated) in the MSD control group and was unmethylated in the MDD group. Arsenic administration resulted in the reduction of methylation at only one site (XhoI), while hypermethylation in the 4.3 and 14.6 mg/kg/day groups at the MspI site was also observed (Table VI
).

View larger version (88K):
[in this window]
[in a new window]
|
Fig. 5. Lane M, bp ladder (from bottom, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 bp). Lane 1, positive control (site known to be methylated in untreated C57BL/6 mice; from Counts et al., 1997). Lane 2, negative control (site known to be unmethylated in untreated C57BL/6 mice). Lane 3, partially methylated control (site known to be partially methylated in untreated C57BL/6 mice). Even numbered lanes represent the amplified products of primer set 962/1487 without prior digestion. Pre-PCR digestion of liver DNA with methylation sensitive enzymes HaeII (Lanes 5 and 7), HpaII (Lanes 13 and 15), Msp (Lanes 17 and 19), AvaII (Lanes 25 and 27), HhaI (Lanes 33 and 35), EcoRII (Lanes 37 and 39) and AluI (Lanes 41 and 43) prevented amplification of region 962/1487. No sites were found to be fully methylated in this region. Heterogeneous methylation was seen in lanes digested by SmaI (Lanes 9 and 11), XhoI (Lanes 21 and 23) and HaeIII (Lanes 29 and 31).
|
|

View larger version (80K):
[in this window]
[in a new window]
|
Fig. 6. Lane M, bp ladder (from bottom, 100, 200, 300, 40, 500, 600, 700, 800, 900, 1000 bp). Lane 1, positive control (site known to be methylated in untreated C57BL/6 mice; from Counts et al., 1997). Lane 2, negative control (site known to be unmethylated in untreated C57BL/6 mice). Lane 3, partially methylated control (site known to be partially methylated in untreated C57BL/6 mice). Even numbered lanes represent the amplified products of primer set 962/1487 without prior digestion. Pre-PCR digestion of liver DNA with methylation sensitive enzymes HaeII (Lanes 5 and 7), SmaI (Lanes 9 and 11), HpaII (Lanes 13 and 15), XhoI (Lanes 31 and 23), HhaI (Lanes 33 and 35), EcoRII (Lanes 37 and 39) and AluI (Lanes 41 and 43) prevented amplification of region 962/1487. No sites were found to be fully methylated in this region. Heterogeneous methylation was seen in lanes digested by MspI (Lanes 17 and 19), AvaII (Lanes 25 and 27) and HaeIII (Lanes 29 and 31).
|
|
Discussion
Sodium arsenite was moderately toxic at all dose levels tested. Histopathological changes in the C57BL/6J mouse liver was observed after administration of a methyl-deficient diet for 130 days. Sodium arsenite increased the incidence and severity of steatosis and microgranulomas in the livers of treated mice.
Steatosis represents a reversible toxic insult to the liver and its occurrence has been documented in rodents receiving a methyl-deficient diet (43). Steatosis is a frequent cellular response to toxicity and its prevalence in the liver is particularly common because it has an important role in lipid metabolism. The mechanism for this pathological change is unclear, but may involve the inhibition of lipid excretion out of the cell. S-adenosyl-methionine is a major intracellular methyl group donor and is critical in the conversion of phosphotidylethanolamine to phosphotidylcholine, an essential component of specific lipoproteins that transport fat out of the liver (51,52). Reductions in available methyl-donors may therefore induce liver toxicity. Arsenic may exacerbate the response by reducing or diverting methyl-donor pools and altering intracellular methylation reactions.
The severity of hepatic fat accumulation was exacerbated in all groups treated with arsenic when compared with the MDD control group. The increase was statistically significant in the 2.6 and 4.3 mg/kg/day groups. An increase in the number of inflammatory microfoci in hepatic cells (microgranulomas) was also apparent in animals receiving a methyl-deficient diet and treated with arsenic although no obvious doseresponse relationship was evident. The greatest severity of hepatic inflammation was seen in the group receiving arsenic at 4.3 mg/kg/day, which was statistically greater than the MDD control group. This lesion is believed to be a consequence of focal necrosis of hepatocytes with an attendant inflammatory reaction (53). The severe hepatic parenchymal damage seen in two arsenic-treated mice is believed to be a consequence of repeated toxic insult and loss of hepatocytes.
Inorganic arsenic metabolism involves several methylation steps. Exposure to arsenic may deplete or divert intracellular methyl donors from inherent cellular functions that require methyl groups to the methylation of arsenic. Given the growing body of evidence suggesting the involvement of aberrant DNA methylation in carcinogenesis, we assessed genomic and Ha-ras methylation status in methyl-deficient arsenic treated mice.
We have determined that methyl-deficiency and sodium arsenite administration reduces genomic DNA methylation. With the exception of the 14.6 mg/kg/day group, a statistically significant response was observed between the concentration of administered arsenic and the hypomethylation of genomic DNA. We speculate that the divergence from the dose-response relationship in the 14.6 mg/kg/day group is the result of a potential overestimation in water consumption due to spontaneous water bottle drip. Animals in the 14.6 mg/kg/day group consumed ~1 g (1 ml) of water/mouse/day compared with mice in other groups, which consumed at least twice this amount (Table VII
). The effect of water bottle drip is pronounced in the 14.6 mg/kg/day group because measurement error increases with reductions in water consumption. Estimated dose in this treatment group was therefore subject to the greatest error and overestimation, and may be responsible for the high dose group not maintaining the doseresponse relationship observed at lower doses.
View this table:
[in this window]
[in a new window]
|
Table VII. Mean water intake and dose arsenic consumed after exposure to varying concentrations of sodium arsenite in drinking water
|
|
Several groups have reported reductions in genomic DNA methylation after the administration of methyl-deficient diets (31,33,5456). Rats fed choline deficient diets develop hepatic lesions that progress through two distinct phases; the first phase is characterized by genomic hypomethylation, severe steatosis and increased cellular turnover; the second phase is distinguished by gradual clearance of deposited fat, fibrosis and parenchymal nodularity. This study confirms that methyl-deficient diets reduce DNA methylation and further establishes arsenic's role in reducing genomic DNA methylation. Chen et al. (57) have validated our findings and report that chronic administration of arsenate or arsenite under methyl-sufficient conditions also reduces hepatic DNA methylation in the mouse. The mechanism by which global methylation patterns are altered by arsenic is unknown. We hypothesize that reductions in DNA methylation are the result of a depletion in the methyl-donor pool by arsenic. Alternatively, arsenic may reduce DNA methylation by inhibiting the DNA methyltransferase. Reductions in the enzymatic activity of the DNA methyltransferase have previously been demonstrated in transformed rat liver cells and may contribute to reductions in DNA methylation (58).
DNA methylation is associated with the regulation of several different processes in mammalian cells including the expression level of tumor promoting genes (29,33,35,55), chromatin structure (59), DNA replication (60), genomic imprinting (61) and somatic X chromosome inactivation in females (62). DNA methylation has a profound effect on DNA structure by facilitating the change from the common B form to the inactive Z form. Methyl groups bound to cytosine have the effect of sterically extending into the major groove of B DNA introducing hydrophobicity to the region and altering the specificity and binding of DNA-associated proteins. Several DNA binding proteins are inhibited when cytosines in its recognition sequences are methylated (63,64). DNA hypomethylation is associated with tumor cell lines, transformed cells and primary human tumors. Whether aberrant methylation is a consequence of tumorigenesis or a causative factor is not apparent (65). In this study, hypomethylation of DNA is a relatively early finding and may suggest causation rather than consequence.
This study further establishes that a methyl-deficient diet and arsenic administration reduces methylation at the 5' regulatory region of Ha-ras. The CpG island amplified by primer set 962/1487 was determined to be primarily unmethylated and is consistent with earlier reports (47). Methyl group deficiency and arsenic had little effect on methylation status in this region. The non-CpG island segment amplified by primer set 474/976 was relatively more methylated than segment 962/1487 and methyl-deficiency and arsenic reduced methylation to a greater extent. Methyl-deficiency and sodium arsenite treatment resulted in reductions in five of the 11 restriction sites examined in segment 474/976 and in one out of four possible sites in segment 962/1487 suggesting that methyl-deficiency and arsenic induces Ha-ras hypomethylation.
Numerous studies have focused on arsenic's role in regulating the methylation and expression of protooncogenes. To date, ~60 genes have been observed to be differentially expressed in hepatic biopsies of arsenic exposed individuals and rat liver cell lines (56,66,67). The genes affected by arsenic include several cyclin genes, E2F3, c-myc and the estrogen receptor. These initial gene expression studies show potentially important aberrant gene expression patterns associated with arsenic-induced malignant transformation and are an apparent prelude to the current study. This investigation has observed arsenic-induced hypomethylation at the 5' regulatory region of Ha-ras in an animal model. Hypomethylation in the promoter region of Ha-ras is important to the extent that cis elements capable of influencing expression are present in that portion of the gene. Reductions in DNA methylation at these regulatory regions may contribute to arsenic induced carcinogenesis by facilitating increased expression and cell cycle dysregulation. Arsenic-induced DNA hypomethylation is a probable mechanism for gene activation and should be investigated further. The results reported here provide a basis for understanding changes in DNA methylation patterns and gene expression at other tumor promoting or tumor suppressing genes that may result from arsenic exposure.
 |
Notes
|
---|
3 To whom correspondence should be addressed Email: jfroines{at}ucla.edu 
 |
Acknowledgments
|
---|
The authors would like to thank Dr Raymond Tice of Integrated Laboratory Systems for his generous insights and the National Institute of Environmental Health Sciences (NIEHS) whose contributions made this study possible. The research reported in this document has been funded in part by the Toxic Substances Research and Teaching Program (TSRTP), Center for Occupational and Environmental Health (COEH), and the National Institute of Environmental Health Services (Grant 5P30ES07048-06).
 |
References
|
---|
-
Chen,C.J., Chuang,Y.C., Lin,T.M. and Wu,H.Y. (1985) Malignant neoplasms among residents of a blackfoot disease-endemic area in Taiwan: high-arsenic artesian well water and cancers. Cancer Res., 45, 58955899.[Abstract]
-
Chen,C.J., Chuang,Y.C., You,S.L., Lin,T.M. and Wu,H.Y. (1986) A retrospective study on malignant neoplasms of bladder, lung and liver in blackfoot disease endemic area in Taiwan. Br. J. Cancer, 53, 399405.[ISI][Medline]
-
Chen,C.J., Chen,C.W., Wu,M.M. and Kuo,T.L. (1992) Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br. J. Cancer, 66, 888892.[ISI][Medline]
-
Chiang,H.S., Guo,H.R., Hong,C.L., Lin,S.M. and Lee,E.F. (1993) The incidence of bladder cancer in the blackfoot disease endemic area in Taiwan. Br. J. Urol., 71, 274278.[ISI][Medline]
-
Hopenhayne-Rich,C., Biggs,M.L., Smith,A.H., Kalman,D.A. and Moore,L.E. (1996) Methylation study of a population environmentally exposed to arsenic in drinking water. Environ. Health Perspect., 104, 620628.[ISI][Medline]
-
Mabuchi,K., Lilienfeld,A.M. and Snell,L.M. (1979) Lung cancers among pesticide workers exposed to inorganic arsenicals. Arch. Environ. Health, 34, 312320.[ISI][Medline]
-
IARC (1980) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 23. IARC, Lyon, 37 pp.
-
Leonard,A. and Lauwerys,R.R. (1980) Carcinogenicity, teratogenicity and mutagenicity of arsenic. Mutat. Res., 75, 4962.[ISI][Medline]
-
Huff,J., Chan,P. and Nyska,A. (2000) Is the human carcinogen arsenic carcinogenic to laboratory animals. Toxicol. Sci., 55, 1723.[Free Full Text]
-
Healy,S.M., Casarez,E.A., Ayala-Fierro,F. and Aposhian,H.V. (1998) Enzymatic methylation of arsenic compounds. V. Arsenite methyltransferase in tissues of mice. Toxicol. Appl. Pharmacol., 148, 6570.[ISI][Medline]
-
Lerman,S.A., Clarkson,T.W. and Gerson,R.J. (1983) Arsenic uptake and metabolism by liver cells is dependent on arsenic oxidation state. Chem.-Biol. Interact., 45, 401406.[ISI][Medline]
-
Feinberg,A.P., Gehrke,C.W., Kuo,K.C. and Ehrlich,M. (1988) Reduced genomic 5-methyl-cytosine in human colonic neoplasia. Cancer Res., 48, 11591161.[Abstract]
-
Jones,P.A. and Buckley,J.D. (1990) The role of DNA methylation in cancer. Adv. Cancer Res., 54, 123.[Medline]
-
Bernardino,J., Roux,C., Almeida,A., et al. (1997) DNA hypomethylation in breast cancer: An independent parameter of tumor progression. Cancer Gene. Cytogenet., 97, 8389.
-
Vorce,R.L. and Goodman,J.I. (1991) Hypomethylation of ras oncogenes in chemically induced and spontaneous B6C3F1 mouse liver tumors. J. Toxicol. Environ. Health, 34, 367384.[ISI][Medline]
-
Ohtani-Fujita,N., Fujita,T., Aoike,A., Osifchin,N.E., Robbins,P.D. and Sakai,T. (1993) CpG methylation inactivates the promoter activity of the human retinoblastoma tumor-suppressor gene. Oncogene, 8, 10631067.[ISI][Medline]
-
Issa,J.P., Vertino,P.M., Wu,J., Sazawai,S., Celano,P., Nelkin,B.D., Hamilton,S.R. and Baylin,S.B. (1993) Increased cytosine DNA-methyltransferase activity during colon cancer progression. J. Natl Cancer Inst., 85, 12351240.[Abstract]
-
Vogelstein,B., Fearon,E.R., Hamilton,S.R., et al. (1988) Genetic alterations during colorectal-tumor development. N. Engl. J. Med., 319, 525532.[Abstract]
-
Gama-Sosa,M.A., Slagel,V.A., Trewyn,R.W., Oxenhandler,R., Kuo,K.C., Gehrke,C.W. and Ehrlich,M. (1983) The 5-methylcytosine content of DNA from human tumors, Nucleic Acid Res., 11, 68836894.
-
Kim,Y., Giuliano,A., Hatch,K.D., Schneider,A., Nour,M.A., Dallal,G.E., Selhub,J. and Mason,J.B. (1994) Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma. Cancer, 74, 893899.[ISI][Medline]
-
Vorce,R.L. and Goodman,J.I. (1989) Altered methylation of ras oncogenes in benzidine-induced B6C3F1 mouse liver tumors. Toxicol. Appl. Pharmacol., 100, 398410.[ISI][Medline]
-
Hoffman,R.M. (1984) Altered methionine metabolism, DNA methylation and oncogene expression in carcinogenesis. Biochim. Biophys. Acta, 738, 4987.[ISI][Medline]
-
Jones,P.A. (1996) DNA methylation errors and cancer. Cancer Res., 56, 24632467.[ISI][Medline]
-
Razin,A. and Kafri,T. (1994) DNA methylation from embryo to adult. Prog. Nucl. Acids Res. Mol. Biol., 48, 5381.[ISI][Medline]
-
Ferguson,A.T., Lapidus,R.G., Baylin,S.B. and Davidson,N.E. (1995) Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression. Cancer Res., 55, 22792283.[Abstract]
-
Counts,J.L. and Goodman,J.I. (1995) Hypomethylation of DNA: a nongenotoxic mechanism involved in tumor promotion. Toxicol. Lett., 82/83, 663672.
-
Maronpot,R.R., Fox,T., Malarkey,D.E. and Goldsworthy,T.L. (1995) Mutation in the ras proto-oncogene: clues to etiology and molecular pathogenesis of mouse liver tumors. Toxicology, 101, 125156.[ISI][Medline]
-
Ray,J.S., Harbison,M.L., McClain,R.M. and Goodman,J.I. (1994) Alterations in the methylation status and expression of the raf oncogene in phenobarbital-induced and spontaneous B6C3F1 mouse liver tumors. Mol. Carcinog., 9, 155166.[ISI][Medline]
-
Wainfan,E., Dizik,M., Stender,M. and Christman,J. (1989) Rapid appearance of hypomethylated DNA in livers of rats fed cancer promoting, methyl-deficient diets. Cancer Res., 49, 40944097.
-
Balaghi,M. and Wagner,C. (1993) DNA methylation in folate deficiency: Use of CpG methylase. Biochem. Biophys. Res. Commun., 193, 11841190.[ISI][Medline]
-
Counts,J.L., Sarmiento,J.I., Harbison,M.L., Downing,J.C., McClain,R.M. and Goodman,J.I. (1996) Cell proliferation and global methylation status changes in mouse liver after phenobarbital and/or choline-devoid, methionine-deficient diet administration. Carcinogenesis, 17, 12511257.[Abstract]
-
Counts,J.L., McClain,R.M. and Goodman,J.I. (1997) Comparison of effect of tumor promoter treatments on DNA methylation status and gene expression in B6C3F1 and C57BL/6 mouse liver and in B6C3F1 mouse liver tumors. Mol. Carcinog., 18, 97106.
-
Dizik,M., Christman,J.K. and Wainfan,E. (1991) Alterations in expression and methylation of specific genes in livers of rats fed a cancer promoting methyl-deficient diet. Carcinogenesis, 12, 13071312.[Abstract]
-
Christman,J.K., Sheikhnejad,G., Dizik,M., Abileah,S. and Wainfan,E. (1993) Reversibility of changes in nucleic acid methylation and gene expression induced in rat liver by severe dietary methyl deficiency. Carcinogenesis, 14, 551557.[Abstract]
-
Wainfan,E. and Poirier,L.A. (1992) Methyl groups in carcinogenesis: Effects on DNA methylation and gene expression. Cancer Res. (Suppl.), 52, 2071s2077s.[Abstract]
-
Hsieh,L.L., Wainfan,E., Hoshina,S., Dizik,M. and Weinstein,I.B. (1989) Altered expression of retrovirus-like sequences and cellular oncogenes in mice fed methyl-deficient diets. Cancer Res., 49, 37953799.[Abstract]
-
Salmon,W.D. and Copeland,D.H. (1954) Liver carcinoma and related lesions in chronic choline deficiency. Ann. NY Acad. Sci., 57, 664677.[ISI]
-
Salmon,W.D., Copeland,D.H. and Burns,M.J. (1955) Hepatomas in choline deficiency. J. Natl. Cancer Inst., 15, 15491568.[ISI]
-
Mikol,Y.B., Hoover,K.L., Creasia,D. and Poirier,L.A. (1983) Hepatocarcinogenesis in rats fed methyl-deficient, amino-acid-defined diets. Carcinogenesis, 4, 16191629.[ISI][Medline]
-
Newberne,P.M. and Rogers,A.E. (1986) Labile methyl groups and the promotion of cancer. Ann. Rev. Nutr., 6, 407432.
-
Newberne,P.M., deCamargo,J.L. and Clark,A.J. (1982) Choline deficiency, partial hepatectomy and liver tumors in rats and mice. Toxicol. Pathol., 2, 95106.
-
Yokoyama,S., Sell,M.A., Reddy,T.V. and Lombardy,B. (1985) Hepatogenesis and promoting action of a choline-devoid diet in the rat. Cancer Res., 45, 28342842.[Abstract]
-
Ghoshal,A.K. and Farber,E. (1984) The induction of liver cancer by dietary deficiency of choline and methionine without added carcinogens. Carcinogenesis, 5, 13671370.[Abstract]
-
Buckley,G.F. and Hartroft,W.S. (1955) Pathology of choline deficiency in the mouse. Arch. Pathol., 59, 185197.[ISI]
-
Henning,S., Swendseid,M. and Coulson,W. (1997) Male rats fed methyl-and folate defined deficient diets with or without niacin develop hepatic carcinomas associated with decreased tissue NAD concentrations and altered poly (ADP-ribose) polymerase activity. J. Nutr., 127, 3036.[Abstract/Free Full Text]
-
Couse,J.F., Davis,V.L., Tally,W.C. and Korach,K.S. (1994) An improved method of genomic DNA extraction for screening transgenic mice. Biotechniques, 17, 10301032.[ISI][Medline]
-
Counts,J.L., Kaznowski,J.M., McClain,R.M. and Goodman,J.I. (1997) 5-methylcytosine is present in the 5' flanking region of Ha-ras in mouse liver and increases with ageing. Int. J. Cancer, 72, 491497.[ISI][Medline]
-
Brown,K., Bailleul,B., Ramsden,M., Fee,F., Krumlauf,R. and Balmain,A. (1988) Isolation and characterization of the 5' flanking region of the mouse c-Harvey-ras gene. Mol. Carcinog., 1, 161170.[ISI][Medline]
-
Neades,R., Betz,N.A., Sheng,X. and Pelling,J.C. (1991) Transient expression of the cloned mouse c-Ha-ras 5' upstream region in transfected primary SENCAR mouse keratinocytes demonstrates its power as a promoter element. Mol. Carcinog., 4, 369375.[ISI][Medline]
-
Hollander, M. and Wolfe, D.A. (1973) Nonparametric Statistical Methods. John Wiley, New York, pp. 120123.
-
Hirata,F., Toyoshima,S., Axelrod,J. and Waxdel,M.J. (1980) Phospholipid methylation: a biochemical signal modulating lymphocyte mitogenesis. Proc. Natl Acad. Sci. USA, 77, 862865.[Abstract]
-
Hirata,F. and Axelrod,J. (1978) Enzymatic methylation of phosphatidylethanolamine increases erythrocyte membrane fluidity. Nature, 275, 219220.[ISI][Medline]
-
Harada,K., Tsuneyama,K., Hiramtsu,K. and Nakayuma,Y. (1999) Significance of CD30-positive lymphocytes in livers in primary biliary cirrhosis. J. Gastroenterol. Hepatol., 14, 11971202.[ISI][Medline]
-
Locker,J., Hutt,S. and Lombardi,B. (1986) DNA methylation and hepatocarcinogenesis in rats fed a choline-devoid diet. Carcinogenesis, 7, 13091312.[Abstract]
-
Christman,J.K., Sheikhnejad,G., Dizik,M., Abileah,S. and Wainfan,E. (1993) Reversibility of changes in nucleic acid methylation and gene expression induced in rat liver by severe dietary methyl deficiency. Carcinogenesis, 4, 551557.
-
Tice,R.R., Yager,J.W. and Crecelius,E. (1997) The effect of choline deficiency on orally administered arsenical urinary excretion kinetics and induced DNA damage in male B6C3F1 mice. Mutat. Res., 386, 315334.[ISI][Medline]
-
Chen,H., Liu,J., Lu,T., Goyer,R.A. and Waalkes,M.P. (2001) Induction of DNA hypomethylation in mouse liver after chronic arsenic exposure. Toxicol. Sci., 60, 76.
-
Zhao,C.Q., Young,M.R., Diwan,B.A., Coogan,T.P. and Waalkes,M.P. (1997) Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression. Proc. Natl Acad. Sci. USA, 94, 1090710912.[Abstract/Free Full Text]
-
Tazi,J. and Bird,A. (1990) Alternative chromatin structure at CpG islands. Cell, 60, 909920.[ISI][Medline]
-
Selig,S., Ariel,M., Goitein,R., Marcus,M. and Cedar,H. (1988) Regulation of mouse satellite DNA replication time. EMBO J., 7, 419426.[Abstract]
-
Li,E., Beard,C. and Jaenisch,R. (1993) Role for DNA methylation in genomic imprinting. Nature, 366, 362365.[ISI][Medline]
-
Monk,M. and Grant,M. (1990) Preferential X-chromosome inactivation, DNA methylation and imprinting. Development (Suppl.), 5562.
-
Zacharias,W., Jaworski,A. and Wells,R.D. (1990) Cytosine methylation enhances Z-DNA formation in vivo. J. Bacteriol., 172, 32783283.[ISI][Medline]
-
Murchie,A.I. and Lilley,D.M. (1989) Base methylation and local DNA helix stability. Effect of the kinetics of cruciform extrusion. J. Mol. Biol., 205, 593602.[ISI][Medline]
-
Antequera,F., Boyes,J. and Bird,A. (1990) High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell, 62, 503514.[ISI][Medline]
-
Chen,H., Liu,J., Merrick,B.A. and Waalkes,M.P. (2001) Genetic events associated with arsenic-induced malignant transformation: application of cDNA microarray technology. Mol. Carcinog., 30, 7987.[ISI][Medline]
-
Lu,T., Liu,J., LeCluyse,E.L., Zhou,Y.S., Cheng,M.L. and Waalkes,M.P. (2001) Application of cDNA microarray to the study of arsenic-induced liver diseases in the population of Guizhou, China. Toxicol. Sci., 59, 185192.[Abstract/Free Full Text]
Received July 13, 2001;
revised January 30, 2002;
accepted February 13, 2002.