DNA Methylation In Liver Research Articles

Quantification of global DNA methylation with infrared fluorescence in liver and muscle tissues of differentially fed boars

Methylation of cytosine residues at CpG sites is involved in various biological processes to control gene regulation and gene expression. Global DNA methylation is changed in different tumors and in cloned animals. Global DNA methylation can be accurately quantified by dot blot analysis with infrared (IR) fluorophores. Methylated lambda DNA was used as model DNA to develop and validate an immunochemical assay with IR fluorescence detection. Two different IR fluorophores were used, one to detect 5-methylcytosine and another to account for DNA loading. A sensitive infrared detection method was established which is suitable for accurate and reproducible quantification of global DNA methylation across a wide dynamic range. This method was subsequently employed to quantify global DNA methylation in liver and in muscle tissues of boars which have received either a control diet or a methyl supplemented diet in an ongoing study. A significant difference in global DNA methylation is indicated in muscle but not in liver tissue between the two groups of boars.

Effects of subchronic cadmium poisoning on DNA methylation in hens

Cadmium is a persistent pollutant that poses a threat to most biological organisms including birds. Although toxicity of cadmium is mainly linked to cancer, the mechanism of its carcinogenic activity remains poorly understood. Since DNA methylation is linked to cancer, we have examined the effect of cadmium on DNA methylation and DNMTs mRNA expression in hen liver and kidney. Sixty 50-day-old hyline-white hens were randomly allocated into 3 equal groups; a control group fed a basal diet, a low-dose group fed the basal diet spiked with 140 mg/kg CdCl 2, and a high-dose group fed the basal diet spiked with 210 mg/kg CdCl 2. After 60 days, liver and kidney samples were analysed for cadmium by FAAS, DNA methylation level by HPLC and DNMTs mRNA levels by semi-quantitative RT-PCR. The DNA methylation levels and the expressions of DNMT1 and DNMT3a mRNA in liver and kidney were significantly elevated by the cadmium treatment but there was no change in the expression of DNMT3b mRNA in the two tissues. The fact that cadmium increases DNA methylation and the expressions of DNMT1 and DNMT3a mRNA in liver and kidney suggests DNA methylation may be involved in the carcinogenic action of cadmium.

Postweaning Dietary Folate Deficiency Provided through Childhood to Puberty Permanently Increases Genomic DNA Methylation in Adult Rat Liver

Folate plays an important role in the pathogenesis of several chronic diseases by its potential ability to modulate DNA methylation. We hypothesized that the postweaning period might be a highly susceptible period to dietary folate intervention for DNA methylation patterning. We determined the effects of timing and duration of dietary folate intervention provided during the postweaning period on genomic DNA methylation in adult rat liver. In study 1, weanling rats were randomized to receive an amino acid-defined diet containing 0 (deficient), 2 (control), or 8 (supplemented) mg folic acid/kg until 8 wk of age, after which all the rats were fed the control diet until 30 wk of age. In study 2, weanling rats were fed the control diet until 8 wk of age and then randomized to receive the diet containing 0, 2, or 8 mg folic acid/kg until 30 wk of age. In study 3, weanling rats were randomized to receive these diets until 30 wk of age. Dietary folate deficiency, but not supplementation, provided during the postweaning period through childhood to puberty significantly increased genomic DNA methylation by 34–48% ( P < 0.04) in rat liver that persisted into adulthood following a return to the control diet at puberty. In contrast, dietary folate deficiency or supplementation continually imposed at weaning or at puberty did not significantly affect genomic DNA methylation in adult rat liver. Our data suggest that early folate nutrition during postnatal development plays an important role in epigenetic programming that can have a permanent effect in adulthood.

P2-139 Effects of dietary zinc supply during pregnancy on foetal liver DNA methylation of the Insulin-like growth factor 2 ( Igf2) gene in C57BL/J6 mice

P2-139 Effects of dietary zinc supply during pregnancy on foetal liver DNA methylation of the Insulin-like growth factor 2 ( Igf2) gene in C57BL/J6 mice

Aberrant DNA methylation and gene expression in livers of newborn mice transplacentally exposed to a hepatocarcinogenic dose of inorganic arsenic

Our prior work showed that brief exposure of pregnant C3H mice to inorganic arsenic-induced hepatocellular carcinoma (HCC) formation in adult male offspring. The current study examined the early hepatic events associated with this oncogenic transformation. Pregnant mice were exposed to a known carcinogenic dose of arsenic (85 ppm) in the drinking water from gestation days 8 to 18. The dams were allowed to give birth and liver samples from newborn males were analyzed for arsenic content, global DNA methylation and aberrant expression of genes relevant to the carcinogenic process. Arsenic content in newborn liver reached 57 ng/g wet weight, indicating arsenic had crossed the placenta, reached the fetal liver and that significant amounts remained after birth. Global methylation status of hepatic DNA was not altered by arsenic in the newborn. However, a significant reduction in methylation occurred globally in GC-rich regions. Microarray and real-time RT-PCR analysis showed that arsenic exposure enhanced expression of genes encoding for glutathione production and caused aberrant expression of genes related to insulin growth factor signaling pathways and cytochrome P450 enzymes. Other expression alterations observed in the arsenic-treated male mouse newborn liver included the overexpression of cdk-inhibitors and stress response genes including increased expression of metallothionein-1 and decreased expression of betaine-homocysteine methyltransferase and thioether S-methyltransferase. Thus, transplacental exposure to arsenic at a hepatocarcinogenic dose induces alterations in DNA methylation and a complex set of aberrant gene expressions in the newborn liver, a target of arsenic carcinogenesis.

Influence of Gamma Irradiation on DNA Methylation in Liver of Male Rats and their Offspring

In the present work, the influence of gamma radiation (3 Gy) on DNA methylation in slowly proliferating liver tissue of adult male rats and their offspring was studied. Total methylation of DNA was determined on the basis of presence of 5-methylcytosine at the inner cytosine of the C-C-G-G sequence by using restriction enzymes, endonuclease Msp I and its isoschisomer Hpa II. It was found that in the liver of control animals, molecular weight distribution of both Msp I and Hpa II digestion products of DNA and the level of DNA methylation were influenced by age mainly between the 3rd and the 7th days of life. At this time, significant difference was found especially in products of cleavage with enzyme Msp I, where an increase in DNA fragments of medium molecular weight (1 - 7 kb) occurred. The changes in the molecular weight distribution of DNA digestion products in the offspring of control rats were accompanied by an increase in the level of total DNA methylation from 29.8% to 67%. In the offspring of irradiated males, the level of DNA methylation was higher in comparison with the offspring of non-irradiated control males, especially on the 3rd day after birth. We suppose that this finding is conjoined with a transgeneration transfer of radiation-induced genome damage.

Irreversible global DNA hypomethylation as a key step in hepatocarcinogenesis induced by dietary methyl deficiency

Dietary methyl group deprivation is now well recognized as a model of hepatocarcinogenesis in rodents. In the present study, we examined the effects of feeding a methyl-deficient diet followed by a methyl-adequate diet on the extent of methylation of liver DNA and on the formation and evolution of altered hepatic foci. Male F344 rats were fed a methyl-deficient diet for 9, 18, 24, and 36 weeks, followed by re-feeding a methyl-adequate diet for a total of 54 weeks. Similar to previous findings, the methyl-deficient diet resulted in decreased levels of S-adenosylmethionine (SAM), SAM/SAH ratios, and global DNA hypomethylation. Feeding the methyl-adequate diet restored the liver SAM levels and SAM/SAH ratios to control levels in all experimental groups. In contrast, re-feeding the complete diet restored DNA methylation to normal level only in the group that had been fed the methyl-deficient diet for 9 weeks; in animals exposed to methyl deprivation longer, the methyl-adequate diet failed to reverse the hypomethylation of DNA. Liver tissue of rats exposed to methyl deficiency for 9, 18, 24, or 36 weeks was characterized by the persistent presence of placental isoform of glutathione- S-transferase (GSTπ)-positive lesions despite re-feeding the methyl-adequate diet. The persistence of altered hepatic foci in liver after withdrawal of methyl-deficient diet serves as an indication of the carcinogenic potential of a methyl-deficient diet. Substitution of the methyl-deficient diet with complete diet failed to prevent the expansion of initiated foci and restore DNA methylation in animals exposed to deficiency for 18, 24, or 36 weeks. The association between DNA hypomethylation and expansion of foci suggests that stable DNA hypomethylation is a promoting factor for clonal expansion of initiated cells. These results provide an experimental evidence and a mechanistic basis by which epigenetic alterations may contribute to the initiation and promotion steps of carcinogenesis.

The Human γ-Globin Gene Promoters Exhibit Markedly Different Patterns of DNA Methylation in Fetal Liver and Adult Erythroid Cells.

Despite intense investigation, the mechanisms by which human γ- to β-globin developmental gene switching occurs have yet to be fully elucidated. Based on studies in many systems, including human clinical trials with 5-Azacytidine and deoxyazacytidine, methylation has been thought to play an important, and more significantly, reversible role in γ-globin gene silencing. One mechanism by which DNA methylation is likely to effect γ-globin gene expression is through site-specific modification of CpG residues in the promoter regions of the γ-globin genes. For example, CpG methylation status has been proposed to mediate the developmentally-specific binding of Sp1 and the stage selector protein (SSP) complex to the proximal γ-globin promoters. We began this study to determine whether there were other CpG residues in the regions of the γ-globin promoters whose methylation status correlated with γ-gene silencing and thus might also serve as “molecular switches” regulating transcription factor binding, local histone acetylation and globin gene expression. To determine the methylation patterns of the γ-globin promoters, we first purified erythroid cells from human fetal liver (FL) and adult bone marrow (BM) using anti-glycophorin magnetic beads. DNA from the purified cells was subjected to bisulfite modification. The regions of the γ-globin promoters were PCR amplified and subcloned into plasmids. Individual plasmids were then sequenced to determine the methylation status of promoter regions. PCR primers were used which allowed determination of methylation status for CpGs at positions −249, −158, −52, −49. +6, +18, and +49 of the G and Aγ globin genes. An additional CpG at +210 was detectable with the Gγ primers. So far we have analyzed Gγ promoters from three FL and three BM samples. Aγ has been analyzed from two FL and three BM samples. An average of 10 clones have been sequenced for each sample. When results for samples within each condition (i.e., Gγ in FL) were combined for analysis, we see the expected increase in methylation of CpG residues in the Gγ promoter from 38% of all sites in the FL to 73% in the adult BM. This difference increases from 30% in FL to 88% adult BM when the CpGs at −158 and +210 are excluded. Combined methylation at these sites only increases from 7 to 21% between FL and BM and thus does not correlate well with changes in gene expression. Looking at the data another way shows a shift from most of the FL clones (76%) having 0 or 1 sites methylated in the Gγ promoter to 78% of the clones having 6,7 or 8 methylated sites in adult cells. While these results fit with the paradigm that methylation is associated with gene silencing, we saw a very different picture for Aγ. Because the promoter regions have nearly identical sequences, are located very close to each other and are similarly regulated, we expected their methylation patterns to be similar. However, for Aγ 13% of promoter CpGs are methylated in the FL cells but this increases to only 22% in adult erythroid cells. Maximal Aγ promoter methylation occurs at the +6 and +18 CpGs which reach only 33 and 36% methylation in adult erythroid cells. 86% of FL Aγ clones are methylated at only 0–2 promoter CpG sites. This does not change at 83% in adult cells. These results indicate differential methylation of the two human γ-globin genes and suggests that simple promoter methylation is not the primary mechanism of γ-globin gene silencing.

Effect of dibromoacetic acid on DNA methylation, glycogen accumulation, and peroxisome proliferation in mouse and rat liver.

Dibromoacetic acid (DBA) is a drinking water disinfection by-product. Its analogs, dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are liver carcinogens in rodents. We evaluated the ability of DBA to cause DNA hypomethylation, glycogen accumulation, and peroxisome proliferation that are activities previously reported for the two other haloacetic acids. Female B6C3F1 mice and male Fischer 344 rats were administered 0, 1,000, and 2,000 mg/l DBA in drinking water. The animals were euthanized after 2, 4, 7, and 28 days of exposure. Dibromoacetic acid caused a dose-dependent and time-dependent decrease of 20%-46% in the 5-methylcytosine content of DNA. Hypomethylation of the c-myc gene was observed in mice after 7 days of DBA exposure. Methylation of 24 CpG sites in the insulin-like growth factor 2 (IGF-II) gene was reduced from 80.2% +/- 9.2% to 18.8% +/- 12.9% by 2,000 mg/l DBA for 28 days. mRNA expression of the c-myc and IGF-II genes in mouse liver was increased by DBA. A dose-dependent increase in the mRNA expression of the c-myc gene was also observed in rats. In both mice and rats, DBA caused dose-dependent accumulation of glycogen and an increase of peroxisomal lauroyl-CoA oxidase activity. Hence, DBA, like DCA and TCA, induced hypomethylation of DNA and of the c-myc and IGF-II genes, increased mRNA expression of both genes, and caused peroxisome proliferation. Again like DCA, DBA also induced glycogen accumulation. These results indicate that DBA shares biochemical and molecular activities in common with DCA and/or TCA, suggesting that it might also be a liver carcinogen.

Dose-dependence, sex- and tissue-specificity, and persistence of radiation-induced genomic DNA methylation changes

Radiation is a well-known genotoxic agent and human carcinogen that gives rise to a variety of long-term effects. Its detrimental influence on cellular function is actively studied nowadays. One of the most analyzed, yet least understood long-term effects of ionizing radiation is transgenerational genomic instability. The inheritance of genomic instability suggests the possible involvement of epigenetic mechanisms, such as changes of the methylation of cytosine residues located within CpG dinucleotides. In the current study we evaluated the dose-dependence of the radiation-induced global genome DNA methylation changes. We also analyzed the effects of acute and chronic high dose (5Gy) exposure on DNA methylation in liver, spleen, and lung tissues of male and female mice and evaluated the possible persistence of the radiation-induced DNA methylation changes. Here we report that radiation-induced DNA methylation changes were sex- and tissue-specific, dose-dependent, and persistent. In parallel we have studied the levels of DNA damage in the exposed tissues. Based on the correlation between the levels of DNA methylation and DNA damage we propose that radiation-induced global genome DNA hypomethylation is DNA repair-related.

Uteroplacental insufficiency alters DNA methylation, one-carbon metabolism, and histone acetylation in IUGR rats.

Uteroplacental insufficiency leads to intrauterine growth retardation (IUGR) and increases the risk of insulin resistance and hypertriglyceridemia in both humans and rats. Postnatal changes in hepatic gene expression characterize the postnatal IUGR rat, despite the transient nature of the initial in utero insult. Phenomena such as DNA methylation and histone acetylation can induce a relatively static reprogramming of gene transcription by altering chromatin infrastructure. We therefore hypothesized that uteroplacental insufficiency persistently affects DNA methylation and histone acetylation in the IUGR rat liver. IUGR rat pups were created by inducing uteroplacental insufficiency through bilateral uterine artery ligation of the pregnant dam on day 19 of gestation. The SssI methyltransferase assay and two-dimensional thin-layer chromatography demonstrated genome-wide DNA hypomethylation in postnatal IUGR liver. To investigate a possible mechanism for this hypomethylation, levels of hepatic metabolites and enzyme mRNAs involved in one-carbon metabolism were measured using HPLC with coulometric electrochemical detection and real-time RT-PCR, respectively. Uteroplacental insufficiency increased IUGR levels of S-adenosylhomocysteine, homocysteine, and methionine in association with decreased mRNA levels of methionine adenosyltransferase and cystathionine-beta-synthase. Western blotting further demonstrated that increased quantities of acetylated histone H3 also characterized the IUGR liver. Increased hepatic levels of S-adenosylhomocysteine can promote DNA hypomethylation, which is often associated with histone hyperacetylation. We speculate that the altered intrauterine milieu associated with uteroplacental insufficiency affects hepatic one-carbon metabolism and subsequent DNA methylation, which thereby alters chromatin dynamics and leads to persistent changes in hepatic gene expression.

438 The influence of LPS and ethanol stimulation on n-nitrosodimethylamine (NDMA) synthesis by transfected HEPG2 cells

The extent of formation of N-nitrosodimethylamine (NDMA) in the stomachs of rats and mice after simultaneous oral administration of [14C]dimethylamine and potassium nitrite was determined by measuring the methylation of liver DNA. With doses of around 1 mg dimethylamine hydrochloride/kg body weight and 50 mg potassium nitrite/kg body weight, 0.8% of the amine was nitrosated on average. The individual fluctuations ranged from 0.2 to 1.3% in the rat and from 0.2 to 1.9% in the mouse. Simultaneous administration of 50 mg sodium ascorbate (vitamin C)/kg body weight inhibited the nitrosation by about 80% while 50 mg α-tocopherol acetate (vitamin E)/kg body weight reduced the nitrosation by about a half. Assuming similar kinetics and conditions of nitrosation in rats and man, a comparison of the formation of NDMA in vivo from dietary dimethylamine and nitrite with the estimated human uptake of preformed NDMA revealed that in vivo formation in the stomach of man is probably negligible.

Aging results in hypermethylation of ribosomal DNA in sperm and liver of male rats

There is a concern that increased paternal age may be associated with altered fertility and an increased incidence of birth defects in man. In previous studies of aged male rats, we have found abnormalities in the fertility and in the embryos sired by older males. Aging in mammals is associated with alterations in the content and patterns of DNA methylation in somatic cells; however, little is known in regard to germ cells. A systematic search for global and gene-specific alterations of DNA methylation in germ cells and liver of male rats was done. Restriction landmark genomic scanning, a method used to determine specific methylation patterns of CpG island sequences, has revealed a region of the ribosomal DNA locus that is preferentially hypermethylated with age in both spermatozoa and liver. In contrast, all single copy CpG island sequences in spermatozoa and in liver remain unaltered with age. We further demonstrate that a large proportion of rat ribosomal DNA is normally methylated and that regional and site-specific differences exist in the patterns of methylation between spermatozoa and liver. We conclude that patterns of ribosomal DNA methylation in spermatozoa are vulnerable to the same age-dependent alterations that we observe in normal aging liver. Failure to maintain normal DNA methylation patterns in male germ cells could be one of the mechanisms underlying age-related abnormalities in fertility and progeny outcome.

Effects of vitamin B12 and folate deficiencies on DNA methylation and carcinogenesis in rat liver.

Deficiencies of the major dietary sources of methyl groups, methionine and choline, lead to the formation of liver cancer in rodents. The most widely investigated hypothesis has been that dietary methyl insufficiency results in abnormal DNA methylation. Vitamin B12 and folate also play important roles in DNA methylation since these two coenzymes are required for the synthesis of methionine and S-adenosyl methionine, the common methyl donor required for the maintenance of methylation patterns in DNA. The aim of this study was to review the effects of methyl-deficient diets on DNA methylation and liver carcinogenesis in rats, and to evaluate the role of vitamin B12 status in defining carcinogenicity of a methyl-deficient diet. Several studies have shown that a methyl-deficient diet influences global DNA methylation. Evidence from in vivo studies has not clearly established a link between vitamin B12 and DNA methylation. We reported that vitamin B12 and low methionine synthase activity were the two determinants of DNA hypomethylation. Choline- or choline/methionine-deficient diets have been shown to cause hepatocellular carcinoma in 20-50% of animals after 12-24 months. In contrast, the effect of vitamin B12 withdrawal, in addition to choline, methionine and folate, induced hepatocellular carcinoma in less than 5% of rats.

Development and application of a sensitive and rapid immunoassay for the quantitation of N7-methyldeoxyguanosine in DNA samples.

N7-Methyldeoxyguanosine (N7-MedG) in DNA is a biomarker of exposure to environmental and endogenous methylating agents and may be of use in epidemiological studies. To quantitate N7-MedG in human samples, a sensitive assay system that uses only small quantities of DNA (<10 microg) is required. To this end, polyclonal antibodies against the imidazole ring-opened form of N7-MedG have been used to develop a highly sensitive immunoslot blot (ISB) assay. The limit of detection of the assay is 0.10 micromol of N7-MedG/mol of deoxyguanosine (dG) using 1 microg of DNA per analysis. The method was optimized using in vitro-methylated calf thymus DNA and then applied to a study of DNA methylation in liver and brain tissues of mice following a single iv dose of the antitumor agent Temozolomide. The amount of N7-MedG in both tissues was strictly proportional to dose over a range of 10-200 mg of Temozolomide/kg of body weight. The ISB assay was then validated using pyloric DNA of rats treated with N-methyl-N'-nitro-N-nitrosoguanidine and DNA samples from human bladder tumors, for both of which N7-MedG levels had already been quantitated by an HPLC/(32)P-postlabeling method previously described. The results showed a high degree of correlation (r = 0.98) between the two assays. The ISB assay was then applied to a range of human samples. A series of peripheral blood mononuclear cell DNA samples from cancer patients following treatment with Temozolomide had levels of N7-MedG ranging from 0.22 to 320 micromol/mol of dG. DNA samples from colon carcinoma and normal colorectal mucosa from individuals not known to be exposed to methylating agents contained levels of 0.11-1.34 micromol of N7-MedG/mol of dG. The ISB assay offers the potential for the rapid and high-throughput analysis of DNA obtained from routine biopsies and blood samples, thus enabling the determination of the extent of human exposure to environmental and endogenous sources of methylating agents in large-scale biomonitoring studies.

Zinc Deficiency in the Juvenile Rat Promoter Dna Methylation and Gene Expression in Liver

Zinc Deficiency in the Juvenile Rat Promoter Dna Methylation and Gene Expression in Liver

Effect of trihalomethanes on cell proliferation and DNA methylation in female B6C3F1 mouse liver.

Trihalomethanes (chloroform, bromodichloromethane, chlorodibromomethane, and bromoform) are regulated organic contaminants in chlorinated drinking water. In female B6C3F1 mouse liver, the 4 trihalomethanes have demonstrated carcinogenic activity when administered by oral gavage; however, chloroform was not carcinogenic when administered in drinking water. Female B6C3F1 mice were administered the trihalomethanes for 11 days by gavage at 2 dose levels or in the drinking water at approximately 75% saturation. When administered by gavage, the trihalomethanes were toxic to the liver, increased the liver:body weight (bw) ratio, and increased the proliferating cell nuclear antigen-labeling index (PCNA-LI). Chloroform and bromodichloromethane were the most toxic, and they increased the liver:bw ratio the most, while bromoform and chloroform increased the PCNA-LI the most. When administered in drinking water, the toxicity of the trihalomethanes was similar to their low gavage-dose. Furthermore, only chloroform significantly increased the liver:bw ratio and bromoform and chloroform increased the PCNA-LI. Chloroform and bromodichloromethane decreased the level of 5-methylcytosine in hepatic DNA. Methylation in the promoter region of the c-myc gene was reduced by the trihalomethanes. Chloroform administered by gavage was more efficacious than given in drinking water; the efficacy of the other trihalomethanes did not differ for the 2 routes. Thus, in mouse liver, the trihalomethanes administered by gavage enhanced cell proliferation and decreased the methylation of the c-myc gene, consistent with their carcinogenic activity. Furthermore, the more modest toxicity, enhancement of cell proliferation, and decreased methylation induced by chloroform administered in drinking water correlated with its lack of carcinogenic activity. Hence, the activity of the trihalomethanes was dependent on the rate of delivery, i.e. rapid by oral gavage and more slowly in drinking water.

P4E108 - Effect of chloroform, haloacetic acids and trichloroethylene on DNA methylation in liver and tumors of female B6C3F1 mice

P4E108 - Effect of chloroform, haloacetic acids and trichloroethylene on DNA methylation in liver and tumors of female B6C3F1 mice

Effect of dichloroacetic acid and trichloroacetic acid on DNA methylation in liver and tumors of female B6C3F1 mice.

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are found in drinking water and are metabolites of trichloroethylene. They are carcinogenic and promote liver tumors in B6C3F1 mice. Hypomethylation of DNA is a proposed nongenotoxic mechanism involved in carcinogenesis and tumor promotion. We determined the effect of DCA and TCA on the level of DNA methylation in mouse liver and tumors. Female B6C3F1 mice 15 days of age were administered 25 mg/kg N-methyl-N-nitrosourea and at 6 weeks started to receive 25 mmol/liter of either DCA or TCA in their drinking water until euthanized 44 weeks later. Other animals not administered MNU were euthanized after 11 days of exposure to either DCA or TCA. DNA was isolated from liver and tumors, and after hydrolysis 5-methylcytosine (5MeC) and the four bases were separated and quantitated by HPLC. In animals exposed to either DCA or TCA for 11 days but not 44 weeks, the level of 5MeC in DNA was decreased in the liver. 5MeC was also decreased in liver tumors from animals exposed to either chloroacetic acid. The level of 5MeC in TCA-promoted carcinomas appeared to be less than in adenomas. Termination of exposure to DCA, but not to TCA, resulted in an increase in the level of 5MeC in adenomas to the level found in noninvolved liver. Thus, hypomethylated DNA was found in DCA and TCA promoted liver tumors and the difference in the response of DNA methylation to termination of exposure appeared to support the hypothesis of different mechanisms for their carcinogenic activity.

Effect of Dichloroacetic Acid and Trichloroacetic Acid on DNA Methylation in Liver and Tumors of Female B6C3F1 Mice

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are found in drinking water and are metabolites of trichloroethylene. They are carcinogenic and promote liver tumors in B6C3F1 mice. Hypomethylation of DNA is a proposed nongenotoxic mechanism involved in carcinogenesis and tumor promotion. We determined the effect of DCA and TCA on the level of DNA methylation in mouse liver and tumors. Female B6C3F1 mice 15 days of age were administered 25 mg/kg N-methyl-N-nitrosourea and at 6 weeks started to receive 25 mmol/liter of either DCA or TCA in their drinking water until euthanized 44 weeks later. Other animals not administered MNU were euthanized after 11 days of exposure to either DCA or TCA. DNA was isolated from liver and tumors, and after hydrolysis 5-methylcytosine (5MeC) and the four bases were separated and quantitated by HPLC. In animals exposed to either DCA or TCA for 11 days but not 44 weeks, the level of 5MeC in DNA was decreased in the liver. 5MeC was also decreased in liver tumors from animals exposed to either chloroacetic acid. The level of 5MeC in TCA-promoted carcinomas appeared to be less than in adenomas. Termination of exposure to DCA, but not to TCA, resulted in an increase in the level of 5MeC in adenomas to the level found in noninvolved liver. Thus, hypomethylated DNA was found in DCA and TCA promoted liver tumors and the difference in the response of DNA methylation to termination of exposure appeared to support the hypothesis of different mechanisms for their carcinogenic activity.