Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum.

Epigenetic mechanisms regulate programmed gene expression during prenatal neurogenesis and serve as a mediator between genetics and environment in postnatal life. The recent discovery of 5-hydroxymethylcytosine (5-hmC), with highest concentration in the brain, has added a new dimension to epigenetic regulation of neurogenesis and the development of complex behavior disorders. Here, we take a candidate gene approach to define the role 5-hmC in Engrailed-2 (EN-2) gene expression in the autism cerebellum. The EN-2 homeobox transcription factor, previously implicated in autism, is essential for normal cerebellar patterning and development. We previously reported EN-2 overexpression associated with promoter DNA hypermethylation in the autism cerebellum but because traditional DNA methylation methodology cannot distinguish 5-methylcytosine (5-mC) from 5-hmC, we now extend our investigation by quantifying global and gene-specific 5-mC and 5-hmC. Globally, 5-hmC was significantly increased in the autism cerebellum and accompanied by increases in the expression of de novo methyltransferases DNMT3A and DNMT3B, ten-eleven translocase genes TET1 and TET3, and in 8-oxo-deoxyguanosine (8-oxo-dG) content, a marker of oxidative DNA damage. Within the EN-2 promoter, there was a significant positive correlation between 5-hmC content and EN-2 gene expression. Based on reports of reduced MeCP2 affinity for 5-hmC, MeCP2 binding studies in the EN-2 promoter revealed a significant decrease in repressive MeCP2 binding that may contribute to the aberrant overexpression of EN-2. Because normal cerebellar development depends on perinatal EN-2 downregulation, the sustained postnatal overexpression suggests that a critical window of cerebellar development may have been missed in some individuals with autism with downstream developmental consequences. Epigenetic regulation of the programmed on-off switches in gene expression that occur at birth and during early brain development warrants further investigation.

Complex epigenetic regulation of engrailed-2 (EN-2) homeobox gene in the autism cerebellum.

The elucidation of epigenetic alterations in the autism brain has potential to provide new insights into the molecular mechanisms underlying abnormal gene expression in this disorder. Given strong evidence that engrailed-2 (EN-2) is a developmentally expressed gene relevant to cerebellar abnormalities and autism, the epigenetic evaluation of this candidate gene was undertaken in 26 case and control post-mortem cerebellar samples. Assessments included global DNA methylation, EN-2 promoter methylation, EN-2 gene expression and EN-2 protein levels. Chromatin immunoprecipitation was used to evaluate trimethylation status of histone H3 lysine 27 (H3K27) associated with gene downregulation and histone H3 lysine 4 (H3K4) associated with gene activation. The results revealed an unusual pattern of global and EN-2 promoter region DNA hypermethylation accompanied by significant increases in EN-2 gene expression and protein levels. Consistent with EN-2 overexpression, histone H3K27 trimethylation mark in the EN-2 promoter was significantly decreased in the autism samples relative to matched controls. Supporting a link between reduced histone H3K27 trimethylation and increased EN-2 gene expression, the mean level of histone H3K4 trimethylation was elevated in the autism cerebellar samples. Together, these results suggest that the normal EN-2 downregulation that signals Purkinje cell maturation during late prenatal and early-postnatal development may not have occurred in some individuals with autism and that the postnatal persistence of EN-2 overexpression may contribute to autism cerebellar abnormalities.

Epigenetic events in tumorigenesis: putting the pieces together.

The development of cancer is a complex multifactorial process traditionally viewed as the stepwise accumulation of genetic alterations. However, recent advances in field of cancer research have established that all major human cancers, in addition to a large number of genetic alterations, exhibit prominent epigenetic abnormalities. This review presents current evidence that epigenetic alterations are not only key features of cancer cells, but they also may be key events in the initiation of carcinogenesis. The early appearance of cancer-linked epigenetic changes that are similar to those found in malignant cells provides a unique opportunity to use them as biomarkers in early cancer detection, indicators of carcinogenic exposure, and in the assessment of the carcinogenic potential of environmental chemical and physical agents.

The role of UDP-glucuronosyltransferases and drug transporters in breast cancer drug resistance.

One of the major limitations of chemotherapy is that often, over time, tumor cells become either inherently resistant or develop multidrug resistance to the treatment. Another limitation of chemotherapy is toxicity to normal tissues and adverse side effects. The reasons for the failure of some cancers to respond to chemotherapeutic drugs are not clear but have been attributed to alterations in many molecular pathways, which include drug metabolizing enzymes and drug transporter genes. Alterations in the energy-dependent ATP-binding cassette (ABC) transporter genes have been suggested to confer a drug-resistant phenotype by decreasing the intracellular accumulation of chemotherapeutic drugs via efflux mechanisms. In addition, polymorphisms in UDP-glucuronosyltransferases (UGTs) have been reported to correlate with clinical outcome and drug resistance. In this review, we provide an overview of known polymorphisms within UGTs and ABC transporter genes that have been reported to have altered expression and/or activity in breast cancer. Those polymorphic variants that affect the clinical efficacy and confer drug resistance of chemotherapeutic agents, including hormonal therapies, taxanes, anthracyclines, and alkylating agents, in breast cancer.

Epigenetic changes in the rat livers induced by pyrazinamide treatment.

Drug-induced liver injury, including drug-induced hepatotoxicity during the treatment of tuberculosis infection, is a major health problem with increasingly significant challenges to modern hepatology. Therefore, the assessment and monitoring of the hepatotoxicity of antituberculosis drugs for prevention of liver injury are great concerns during disease treatment. The recently emerged data showing the ability of toxicants, including pharmaceutical agents, to alter cellular epigenetic status, open a unique opportunity for early detection of drug hepatotoxicity. Here we report that treatment of male Wistar rats with antituberculosis drug pyrazinamide at doses of 250, 500 or 1000 mg/kg/day body weight for 45 days leads to an early and sustained decrease in cytosine DNA methylation, progressive hypomethylation of long interspersed nucleotide elements (LINE-1), and aberrant promoter hypermethylation of placental form glutathione-S-transferase (GSTP) and p16(INK4A) genes in livers of pyrazinamide-treated rats, while serum levels of bilirubin and activity of aminotransferases changed modestly. The early occurrence of these epigenetic alterations and their association with progression of liver injury specific pathological changes indicate that alterations in DNA methylation may be useful predictive markers for the assessment of drug hepatotoxicity.

The impact of tumor growth on plasma homocysteine levels and tissue-specific DNA methylation in Walker-256 tumor-bearing rats.

BACKGROUND: The elevation in plasma homocysteine (Hcy) concentrations has been associated with several types of human cancer; however, the major unanswered question whether or not hyperhomocysteinemia is associated with cancer pathogenesis and is an indicator of tumorigenesis, remains elusive. AIM: To define the impact of tumor growth on the levels of plasma Hcy and to elucidate the underlying mechanisms related to the tumor-associated hyperhomocysteinemia. MATERIALS AND METHODS: Female Wistar rats were inoculated subcutaneously with Walker-256 mammary carcinoma cells. The dynamic of tumor growth, the concentrations of plasma Hcy, and status of DNA methylation in the livers and tumors in tumor-bearing rats were determined. RESULTS: The results of our study demonstrated that development and progression of Walker-256 tumors is associated with both progressive hyperhomocysteinemia and tumor-specific genomic hypomethylation. The pattern of changes in the plasma Hcy concentrations was consistent with linear increase in DNA hypomethylation in tumors and with expansion of Walker-256 tumors. There was significant correlation of the concentrations of plasma Hcy with both parameters (r = 0.73 and r = 0.88, respectively; p 0.05). CONCLUSION: The results of the study provided evidence that growth of Walker-256 tumors is associated with the increased levels of plasma Hcy. More importantly, these findings suggest that an underlying cause of hyperhomocysteinemia in tumor-bearing rats is related to the altered cellular methylation reactions in tumor cells and to tumor proliferation rate, and may serve as metabolic biomarker of cancer.

MicroRNAs in normal and cancer cells: a new class of gene expression regulators.

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at posttranscriptional level. They are involved in cellular development, differentiation, proliferation and apoptosis and play a significant role in cancer. This review describes miRNA biogenesis, their functions in normal cells, and alterations of miRNA sets in cancer and roles of antitumorigenic and oncogenic miRNAs in cancer development.

Age-related effects of methionine-enriched diet on plasma homocysteine concentration and methylation of hepatic DNA in rats.

In the present study we determined the age-related effect of methionine-enriched diet, a model of hyperhomocysteinemia, on the level of plasma homocysteine and hepatic global DNA methylation in rats. Feeding methionine diet to middle-aged rats for only 14 days resulted in a significant increase in plasma homocysteine level and DNA hypomethylation. In contrast, feeding the methionine-containing diet for 2 weeks to juvenile or post-pubertal animals did not alter the level of plasma homocysteine or hepatic DNA methylation. Supplementation of the methionine-enriched diet with vitamins B6, B12 and folic acid prevented both hepatic DNA hypomethylation and an increase of plasma homocysteine concentration in the middle-aged rats. These findings indicate that the elevated level of plasma homocysteine may be indicative of much broader and deeper alterations in intracellular methylation dysfunction, and suggest that dietary enrichment with B-vitamins is essential for the metabolism of homocysteine, especially in adult animals.

Reduction of p53 gene expression in human primary hepatocellular carcinoma is associated with promoter region methylation without coding region mutation.

Functional inactivation of tumor suppressor genes during tumor progression has been shown to occur by either coding region mutation or promoter region methylation. Because of the functional equivalence of these two mechanisms, loss of tumor suppressor function generally occurs by one or the other mechanism, but rarely by both. Aberrant de novo methylation in most tumor suppressor promoter regions is found within CpG islands that occur near the transcription start site. The p53 promoter region is unique in that it does not contain a CpG island and therefore it is possible that methylation at critical CpG sites may be more important in gene silencing than total CpG methylation density. Other than site-specific aflatoxin B(1)-induced mutations, p53 coding region mutations are not frequently observed in most human primary hepatocellular carcinomas. In the present study, paired samples of human primary liver carcinoma and uninvolved tissue obtained from the same individual were evaluated for site-specific p53 promoter methylation status by methylation sensitive single nucleotide primer extension (Ms-SNuPE) and also for coding region mutations using polymerase chain reaction (PCR)- single strand conformation polymorphism (SSCP). The methylation pattern in the uninvolved tissue was variable at specific CpG sites, whereas the same sites had become highly methylated in tumor tissue from the same individual. Associated with de novo methylation, the level of p53 mRNA was significantly reduced in the tumor DNA relative to the uninvolved tissue DNA. None of the samples exhibited coding region mutations. Given that p53 mutations are rare in primary human liver tumors, these data suggest that transcriptional repression by p53 promoter methylation may contribute to tumor progression.

Intracellular S-adenosylhomocysteine concentrations predict global DNA hypomethylation in tissues of methyl-deficient cystathionine beta-synthase heterozygous mice.

Because S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are the substrate and product of essential methyltransferase reactions; the ratio of SAM:SAH is frequently used as an indicator of cellular methylation potential. However, it is not clear from the ratio whether substrate insufficiency, product inhibition or both are required to negatively affect cellular methylation capacity. A combined genetic and dietary approach was used to modulate intracellular concentrations of SAM and SAH. Wild-type (WT) or heterozygous cystathionine beta-synthase (CBS +/-) mice consumed a control or methyl-deficient diet for 24 wk. The independent and combined effect of genotype and diet on SAM, SAH and the SAM:SAH ratio were assessed in liver, kidney, brain and testes and were correlated with relative changes in tissue-specific global DNA methylation. The combined results from the different tissues indicated that a decrease in SAM alone was not sufficient to affect DNA methylation in this model, whereas an increase in SAH, either alone or associated with a decrease in SAM, was most consistently associated with DNA hypomethylation. A decrease in SAM:SAH ratio was predictive of reduced methylation capacity only when associated with an increase in SAH; a decrease in the SAM:SAH ratio due to SAM depletion alone was not sufficient to affect DNA methylation in this model. Plasma homocysteine levels were positively correlated with intracellular SAH levels in all tissues except kidney. These results support the possibility that plasma SAH concentrations may provide a sensitive biomarker for cellular methylation status.

Abnormal folate metabolism and genetic polymorphism of the folate pathway in a child with Down syndrome and neural tube defect.

The association of neural tube defects (NTDs) with Down syndrome (trisomy 21) and altered folate metabolism in both mother and affected offspring provide a unique opportunity for insight into the etiologic role of folate deficiency in these congenital anomalies. We describe here the case of a male child with trisomy 21, cervical meningomyelocele, agenesis of corpus callosum, hydrocephaly, cerebellar herniation into the foramen magnum, and shallow posterior cranial fossa. Molecular analysis of the methylenetetrahydrofolate (MTHFR) gene revealed homozygosity for the mutant 677C-->T polymorphism in both the mother and child. The plasma homocysteine of the mother was highly elevated at 25.0 micromol/L and was associated with a low methionine level of 22.1 micromol/L. Her S-adenosylhomocysteine (SAH) level was three times that of reference normal women, resulting in a markedly reduced ratio of S-adenosylmethionine (SAM) to SAH and significant DNA hypomethylation in lymphocytes. The child had low plasma levels of both homocysteine and methionine and a reduced SAM/SAH ratio that was also associated with lymphocyte DNA hypomethylation. In addition, the child had a five-fold increase in cystathionine level relative to normal children, consistent with over-expression of the cystathionine beta synthase gene present on chromosome 21. We suggest that altered folate status plus homozygous mutation in the MTHFR gene in the mother could promote chromosomal instability and meiotic non-disjunction resulting in trisomy 21. Altered folate status and homozygous TT mutation in the MTHFR gene in both mother and child would be expected to increase the risk of neural tube defects. The presence of both trisomy 21 and postclosure NTD in the same child supports the need for an extended periconceptional period of maternal folate supplementation to achieve greater preventive effects for both NTD and trisomy 21.

Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition.

Hyperhomocysteinemia, a risk factor for cardiovascular disease, is caused by nutritional and/or genetic disruptions in homocysteine metabolism. The most common genetic cause of hyperhomocysteinemia is the 677C-->T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. This variant, with mild enzymatic deficiency, is associated with an increased risk for neural tube defects and pregnancy complications and with a decreased risk for colon cancer and leukemia. Although many studies have reported that this variant is also a risk factor for vascular disease, this area of investigation is still controversial. Severe MTHFR deficiency results in homocystinuria, an inborn error of metabolism with neurological and vascular complications. To investigate the in vivo pathogenetic mechanisms of MTHFR deficiency, we generated mice with a knockout of MTHFR: Plasma total homocysteine levels in heterozygous and homozygous knockout mice are 1.6- and 10-fold higher than those in wild-type littermates, respectively. Both heterozygous and homozygous knockouts have either significantly decreased S-adenosylmethionine levels or significantly increased S-adenosylhomocysteine levels, or both, with global DNA hypomethylation. The heterozygous knockout mice appear normal, whereas the homozygotes are smaller and show developmental retardation with cerebellar pathology. Abnormal lipid deposition in the proximal portion of the aorta was observed in older heterozygotes and homozygotes, alluding to an atherogenic effect of hyperhomocysteinemia in these mice.

Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation.

S-Adenosylmethionine and S-adenosylhomocysteine (SAH), as the substrate and product of essential cellular methyltransferase reactions, are important metabolic indicators of cellular methylation status. Chronic elevation of SAH, secondary to the homocysteine-mediated reversal of the SAH hydrolase reaction, reduces methylation of DNA, RNA, proteins, and phospholipids. High affinity binding of SAH to the active site of cellular methyltransferases results in product inhibition of the enzyme. Using a sensitive new high pressure liquid chromatography method with coulometric electrochemical detection, plasma SAH levels in healthy young women were found to increase linearly with mild elevation in homocysteine levels (r = 0.73; p < 0.001); however, S-adenosylmethionine levels were not affected. Plasma SAH levels were positively correlated with intracellular lymphocyte SAH levels (r = 0.81; p < 0.001) and also with lymphocyte DNA hypomethylation (r = 0.74, p < 0.001). These results suggest that chronic elevation in plasma homocysteine levels, such as those associated with nutritional deficiencies or genetic polymorphisms in the folate pathway, may have an indirect and negative effect on cellular methylation reactions through a concomitant increase in intracellular SAH levels.

Single-site methylation within the p53 promoter region reduces gene expression in a reporter gene construct: possible in vivo relevance during tumorigenesis.

It is not known whether transcriptional suppression by de novo methylation occurs within the promoter region of the p53 gene during multistage tumorigenesis. To address this question, in vivo alterations in the CpG methylation within the rat p53 promoter region were evaluated in control, preneoplastic, and tumor tissue during tumor progression using the folate/methyl-deficient model of hepatocarcinogenesis. Alterations in CpG methylation were found to be site-specific and to vary depending on the stage of carcinogenesis. To further explore the effect of site-specific methylation on p53 promoter activity, reporter gene constructs were prepared containing specifically methylated sites within the p53 promoter region, and the transcriptional activity in cultured mammalian cells was determined in a transient transfection assay. Relative to the unmethylated construct as a positive control, single-site methylation at nucleotide (nt) -450, which occurs 216 nt upstream from the 85-nt minimal promoter region, suppressed promoter activity by 85%. In contrast, single-site methylation at nt -179, which occurs within the minimal essential promoter region, suppressed activity by only 20%. The p53 promoter constructs containing the singly methylated CpG site at nt -450 were then reevaluated for processive changes in methylation status 48 h after transfection, during maximum suppression of promoter activity. Restriction analysis with methylation-sensitive enzymes revealed that de novo methylation had occurred after transfection at previously unmethylated sites. These findings suggest that nt -450 may constitute a critical site for initiation of de novo methylation and processive spreading of methylation associated with transcriptional inactivation of the p53 gene. Furthermore, the results suggest a possible alternative mechanism for the silencing of the p53 gene in tumors that do not have p53 mutations.

Measurement of plasma and intracellular S-adenosylmethionine and S-adenosylhomocysteine utilizing coulometric electrochemical detection: alterations with plasma homocysteine and pyridoxal 5'-phosphate concentrations.

BACKGROUND: The relative changes in plasma and intracellular concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) may be important predictors of cellular methylation potential and metabolic alterations associated with specific genetic polymorphisms and/or nutritional deficiencies. Because these metabolites are present in nanomolar concentrations in plasma, methods of detection generally require time-consuming precolumn processing or metabolite derivatization. METHODS: We used HPLC with coulometric electrochemical detection for the simultaneous measurement of SAM and SAH in 200 microL of plasma, 10(6) lymphocytes, or 10 mg of tissue. Filtered trichloroacetic acid extracts were injected directly into the HPLC system without additional processing and were eluted isocratically. RESULTS: The limits of detection were 200 fmol/L for SAM and 40 fmol/L SAH. In plasma extracts, the interassay CV was 3.4-5.5% and the intraassay CV was 2.8-5.6%. The analytical recoveries were 96.8% and 97.3% for SAM and SAH, respectively. In a cohort of healthy adult women with mean total homocysteine concentrations of 7.3 micromol/L, the mean plasma value was 156 nmol/L for SAM and 20 nmol/L for SAH. In women with increased homocysteine concentrations (mean, 12.1 micromol/L), plasma SAH, but not SAM, was increased (P <0.001), and plasma pyridoxal 5'-phosphate concentrations were reduced (P <0.001). Plasma SAM/SAH ratios were inversely correlated with homocysteine concentrations (r = 0.73; P <0.01), and the SAM/SAH ratio in plasma was directly correlated with the intracellular SAM/SAH ratio in lymphocytes (r = 0.70; P <0.01). CONCLUSIONS: Increased homocysteine in serum is associated with an increase in SAH and a decrease in the SAM/SAH ratio that could negatively affect cellular methylation potential. Accurate and sensitive detection of these essential metabolites in plasma and in specific tissues should provide new insights into the regulation of one-carbon metabolism under different nutritional and pathologic conditions.

Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome.

BACKGROUND: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age. OBJECTIVE: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677C-->T) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers. DESIGN: The frequency of the MTHFR 677C-->T mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status. RESULTS: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677C-->T mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03). CONCLUSION: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.

Uracil misincorporation, DNA strand breaks, and gene amplification are associated with tumorigenic cell transformation in folate deficient/repleted Chinese hamster ovary cells.

Clinical and experimental evidence has linked nutritional folic acid status to both anti- and procarcinogenic activity. Folate supplementation of normal cells appears to have a protective effect; however, folate supplementation of initiated cells may promote neoplastic progression. Given these considerations, the present series of experiments examines alterations in DNA metabolism and cumulative DNA lesions using an in vitro model of folate deprivation and repletion. DNA repair-deficient CHO-UV5 cells were cultured in Ham's F-12 medium or in custom-prepared Ham's F-12 medium lacking in folic acid, thymidine and hypoxanthine for a period of 18 days without cell passage. The results indicated that progressive folate and nucleotide depletion leads to a significant increase in the ratio of dUTP/dTTP and to the misincorporation of uracil into DNA. These alterations were accompanied by growth inhibition, DNA strand breaks, abasic sites and phenotypic abnormalities. After 14 days in culture, there was significant increase in gene amplification potential in the chronically folate-deficient cells, but no significant increase in anchorage-independent growth or in neoplastic transformation. Acute folate repletion of the deficient cells was used as a proliferative stimulus under conditions of dNTP pool imbalance and multiple lesions in DNA. A further increase in gene amplification was accompanied by anchorage-independent growth and neoplastic cell transformation as evidenced by aggressive tumor growth in Balb/c nu/nu mice. Using a sensitive in vitro model system, these results emphasize the essentiality of folic acid for de novo nucleotide synthesis and the integrity of the DNA. However, the in vivo relevance, especially in terms of tumorigenic potential, is not clear.

Hypomethylation of the rat glutathione S-transferase pi (GSTP) promoter region isolated from methyl-deficient livers and GSTP-positive liver neoplasms.

DNA methylation at the 5-position on the cytosine ring in CpG dinucleotides (CpG sites) appears to play an important role in regulating gene expression. In general, there is an inverse relationship between promoter CpG site methylation and the potential for transcription. Thus, changes in DNA methylation density may lead to altered levels of proteins such as glutathione S-transferase pi (GSTP), which is frequently used as a marker to detect hepatocellular foci and neoplasms in the rat. In the present study, the level of CpG methylation in the rat GSTP promoter region was determined in bisulfite-treated DNA isolated from control (untreated) rat livers, chemically induced, GSTP-positive rat liver neoplasms, and methyl-deficient rat livers that contained numerous GSTP-positive foci after administration of a defined diet deficient in folate and choline and low in methionine (0.18%). Eight cytosines between -235 and + 140 in the GSTP promoter region were methylated in a site-specific manner in GSTP-negative control liver, whereas these same sites were hypomethylated in all four chemically-induced, GSTP-positive neoplasms. Similarly, all CpG sites were unmethylated in methyl-deficient liver DNA within 3 weeks of the rats receiving the methyl-deficient diet, and they remained unmethylated throughout the 36-week treatment period. Five of the eight CpG sites are located within consensus sequences for the DNA binding proteins Spl and E2F. This indicates at least one possible mechanism that could potentially lead to transcriptional activation of GSTP in hepatocellular foci and neoplasms during rat hepatocarcinogenesis. These findings suggest that methylation of critical cytosines within the promoter region rather than all CpG-associated cytosines may be a determining factor in regulation of GSTP expression.

Moderate folate depletion increases plasma homocysteine and decreases lymphocyte DNA methylation in postmenopausal women.

To determine the human folate requirement on the basis of changes in biochemical pathways, we studied the effect of controlled folate intakes on plasma homocysteine and lymphocyte DNA methylation and deoxynucleotide content in healthy postmenopausal women. Eight women (49-63 y of age) were housed in a metabolic unit and fed a low folate diet containing 56 microg/d of folate for 91 d. Folate intake was varied by supplementing 55-460 microg/d of folic acid (pteroylglutamic acid) to the diet to provide total folate intake periods of 5 wk at 56 microg/d, 4 wk at 111 microg/d and 3 wk at 286-516 microg/d. A subclinical folate deficiency with decreased plasma folate was created during the first two periods. This resulted in significantly elevated plasma homocysteine and urinary malondialdehyde, and lymphocyte DNA hypomethylation. The folate depletion also resulted in an increased ratio of dUTP/dTTP in mitogen-stimulated lymphocyte DNA and decreased lymphocyte NAD, changes suggesting misincorporation of uracil into DNA and increased DNA repair activity. The DNA hypomethylation was reversed with 286-516 microg/d of folate repletion, whereas the elevated homocysteine decreased with 516 but not 286 microg/d of folate. The results indicate that marginal folate deficiency may alter DNA composition and that the current RDA of 180 microg/d may not be sufficient to maintain low plasma homocysteine concentrations of some postmenopausal women.

Presence and consequence of uracil in preneoplastic DNA from folate/methyl-deficient rats.

Uracil can arise in DNA by misincorporation of dUTP into nascent DNA and/or by cytosine deamination in established DNA. Based on recent findings, both pathways appear to be promoted in the methyl-deficient model of hepatocarcinogenesis. A chronic increase in the ratio dUTP:dTTP with folate/methyl deficiency can result in a futile cycle of excision and reiterative uracil misincorporation leading to premutagenic apyrimidinic (AP) sites, DNA strand breaks, DNA fragmentation and apoptotic cell death. The progressive accumulation of unmethylated cytosines with chronic methyl deficiency will increase the potential for cytosine deamination to uracil and further stress uracil mismatch repair mechanisms. Uracil is removed by a highly specific uracil-DNA glycosylase (UDG) leaving an AP site that is subsequently repaired by sequential action of AP endonuclease, 5'-phosphodiesterase, a DNA polymerase and DNA ligase. Since the DNA polymerases cannot distinguish between dUTP and dTTP, an increase in dUTP:dTTP ratio will promote uracil misincorporation during both DNA replication and repair synthesis. The misincorporation of uracil for thymine (5-methyluracil) may constitute a genetically significant form of DNA hypomethylation distinct from cytosine hypomethylation. In the present study a significant increase in the level of uracil in liver DNA as early as 3 weeks after initiation of folate/methyl deficiency was accompanied by parallel increases in DNA strand breaks, AP sites and increased levels of AP endonuclease mRNA. In addition, uracil was also detected within the p53 gene sequence using UDG PCR techniques. Increased levels of uracil in DNA implies that the capacity for uracil base excision repair is exceeded with chronic folate/methyl deficiency. It is possible that enzyme-induced extrahelical bases, AP sites and DNA strand breaks interact to negatively affect the stability of the DNA helix and stress the structural limits of permissible uracil base excision repair activity. Thus substitution of uracil for thymine induces repair-related premutagenic lesions and a novel form of DNA hypomethylation that may relate to tumor promotion in the methyl-deficient model of hepatocarcinogenesis.