Effect of titanium dioxide nanoparticles on histone modifications and histone modifying enzymes expression in human cell lines.

Titanium dioxide (TiO2) nanoparticles are widely manufactured, with a range of applications in consumer products. Significant toxicity of TiO2 nanoparticles has, however, been recognized, suggesting considerable risk to human health. To evaluate fully their toxicity, assessment of the epigenetic action of these nanoparticles is critical. However, only few studies are available examining the capability of nanoparticles to alter epigenetic integrity. In the present study, the effect of TiO2 nanoparticles exposure on histone modifications, a major epigenetic mechanism, was investigated in human colorectal (Caco-2) and lung (NL20) epithelial cell lines. Histone H3 and H4 modifications were assessed by array analysis using the EpiQuickTM Histone H3 or H4 Modification Multiplex Assay. Seventeen histone modifications were identified with altered levels after exposure to TiO2 nanoparticles. Changes in several selected histone modifications (Caco-2 cells: H3cit, H3K9me3, H3K27me3, H3K36me3, H3K9ac, and H4K8ac; NL20 cells: H3K4me3, H3K9me3, H3K27me3, H3K9ac, and H3K18ac) were verified by Western blot analysis. The results also revealed aberrant expression of histone modifying enzymes in TiO2 exposed cells. Expression levels were determined by array analysis using the Human Epigenetic Chromatin Modification Enzymes RT2 Profiler™ PCR Array, with 12 genes identified in both Caco-2 cells and NL20 cells. qRT-PCR analysis confirmed the array results for several selected histone modifying enzyme genes (ASH1L, CARM1, EHMT2, HAT1, HDAC9, KMT2E, NCOA1, SETDB2, and USP16). The findings from this study clearly demonstrate the impact of TiO2 nanoparticles exposure on histone modification in two human cell lines, supporting potential involvement of this epigenetic mechanism in the toxicity of TiO2 nanoparticles. Hence, for complete assessment of potential risk from nanoparticle exposure, epigenetic studies are critical.

Epigenetic Effects of Nanomaterials and Nanoparticles.

The rise of nanotechnology and widespread use of engineered nanomaterials in everyday human life has led to concerns regarding their potential effect on human health. Adverse effects of nanomaterials and nanoparticles on various molecular and cellular alterations have been well-studied. In contrast, the role of epigenetic alterations in their toxicity remains relatively unexplored. This review summarizes current evidence of alterations in cytosine DNA methylation and histone modifications in response to nanomaterials and nanoparticles exposures in vivo and in vitro. This review also highlights existing knowledge gaps regarding the role of epigenetic alterations in nanomaterials and nanoparticles toxicity. Additionally, the role of epigenetic changes as potential translational biomarkers for detecting adverse effects of nanomaterials and nanoparticles is discussed.

Effect of titanium dioxide nanoparticles on DNA methylation in multiple human cell lines.

Nanoscale titanium dioxide (TiO2) is manufactured in wide scale, with a range of applications in consumer products. Significant toxicity of TiO2 nanoparticles has, however, been recognized, suggesting considerable risk to human health. To evaluate fully their toxicity, assessment of the epigenetic action of these nanoparticles is critical. However, only few studies are available examining capability of nanoparticles to alter epigenetic integrity. In the present study, the effect of TiO2 nanoparticles exposure on DNA methylation, a major epigenetic mechanism, was investigated in in vitro cellular model systems. A panel of cells relevant to portals of human exposure (Caco-2 (colorectal), HepG2 (liver), NL20 (lung), and A-431 (skin)) was exposed to TiO2 nanoparticles to assess effects on global methylation, gene-specific methylation, and expression levels of DNA methyltransferases, MBD2, and UHRF1. Global methylation was determined by enzyme-linked immunosorbent assay-based immunochemical analysis. Degree of promoter methylation across a defined panel of genes was evaluated using EpiTect Methyl II Signature PCR System Array technology. Expression of DNMT1, DNMT3a, DNMT3b, MBD2, and URHF1 was quantified by qRT-PCR. Decrease in global DNA methylation in cell lines Caco-2, HepG2, and A-431 exposed to TiO2 nanoparticles was shown. Across four cell lines, eight genes (CDKN1A, DNAJC15, GADD45A, GDF15, INSIG1, SCARA3, TP53, and BNIP3) were identified in which promotors were methylated after exposure. Altered expression of these genes is associated with disease etiology. The results also revealed aberrant expression of epigenetic regulatory genes involved in DNA methylation (DNMT1, DNMT3a, DNMT3b, MBD2, and UHRF1) in TiO2 exposed cells, which was cell type dependent. Findings from this study clearly demonstrate the impact of TiO2 nanoparticles exposure on DNA methylation in multiple cell types, supporting potential involvement of this epigenetic mechanism in the toxicity of TiO2 nanoparticles. Hence for complete assessment of potential risk from nanoparticle exposure, epigenetic studies are critical.

Epigenetically mediated inhibition of S-adenosylhomocysteine hydrolase and the associated dysregulation of 1-carbon metabolism in nonalcoholic steatohepatitis and hepatocellular carcinoma.

The substantial rise in the prevalence of nonalcoholic steatohepatitis (NASH), an advanced form of nonalcoholic fatty liver disease, and the strong association between NASH and the development of hepatocellular carcinoma indicate the urgent need for a better understanding of the underlying mechanisms. In the present study, by using the Stelic animal model of NASH and NASH-derived liver carcinogenesis, we investigated the role of the folate-dependent 1-carbon metabolism in the pathogenesis of NASH. We demonstrated that advanced NASH and NASH-related liver carcinogenesis are characterized by a significant dysregulation of 1-carbon homeostasis, with diminished expression of key 1-carbon metabolism genes, especially a marked inhibition of the S-adenosylhomocysteine hydrolase ( Ahcy) gene and an increased level of S-adenosyl-l-homocysteine (SAH). The reduction in Ahcy expression was associated with gene-specific cytosine DNA hypermethylation and enrichment of the gene promoter by trimethylated histone H3 lysine 27 and deacetylated histone H4 lysine 16, 2 main transcription-inhibiting markers. These results indicate that epigenetically mediated inhibition of Ahcy expression may be a driving force in causing SAH elevation and subsequent downstream disturbances in transsulfuration and transmethylation pathways during the development and progression of NASH.-Pogribny, I. P., Dreval, K., Kindrat, I., Melnyk, S., Jimenez, L., de Conti, A., Tryndyak, V., Pogribna, M., Ortega, J. F., James, S. J., Rusyn, I., Beland, F. A. Epigenetically mediated inhibition of S-adenosylhomocysteine hydrolase and the associated dysregulation of 1-carbon metabolism in nonalcoholic steatohepatitis and hepatocellular carcinoma.

In vitro analysis of factors influencing CYP1A2 expression as potential determinants of interindividual variation.

Individual differences in drug metabolism contribute to interindividual variation that characterizes responses to drugs and risk in exposure to foreign chemicals. Large individual differences are found in expression levels of CYP1A2, a major drug-metabolizing enzyme. Underlying causes for this variation are not well understood. Several factors, including tobacco smoking, consumption of cruciferous vegetables, and sex, have been associated with modulating CYP1A2 expression. To understand factors regulating expression of CYP1A2 in establishing a causal relationship, this study examined effects of cigarette smoke condensate (CSC), indole-3-carbinol (I3C), and 17ß-estradiol (estradiol) on CYP1A2 expression in in vitro systems using human liver and lung cells. Treatment with CSC (2-25 µg/mL) significantly increased levels of CYP1A2 in six cell lines examined, in a concentration- and time-dependent manner. Fold changes in expression levels relative to controls varied among cell lines. CYP1A2 enzymatic activity also increased with CSC exposure. Treatment of H1299 and HepB3 cells with dietary agent I3C (50 and 100 µmol/L) increased CYP1A2 expression. In human cell lines H1299 and H1395, treatment with estradiol (10 and 100 nmol/L) significantly reduced expression of CYP1A2. Using ChIP assays, effects of CSC on histone modifications were analyzed. Increases in H3K4me3 and H4K16ac were observed at several segments in the CYP1A2 gene, whereas H3K27me3 decreased, following CSC treatment. These results suggest that CYP1A2 expression is affected epigenetically by CSC. Additional studies will be needed to further establish regulatory mechanisms underlying effects of various environmental, dietary, and endogenous factors on CYP1A2 expression in better predicting individual variation.

Suppressing activity of tributyrin on hepatocarcinogenesis is associated with inhibiting the p53-CRM1 interaction and changing the cellular compartmentalization of p53 protein.

Hepatocellular carcinoma (HCC), an aggressive and the fastest growing life-threatening cancer worldwide, is often diagnosed at intermediate or advanced stages of the disease, which substantially limits therapeutic approaches for its successful treatment. This indicates that the prevention of hepatocarcinogenesis is probably the most promising approach to reduce both the HCC incidence and cancer-related mortality. In previous studies, we demonstrated a potent chemopreventive effect of tributyrin, a butyric acid prodrug, on experimental hepatocarcinogenesis. The cancer-inhibitory effect of tributyrin was linked to the suppression of sustained cell proliferation and induction of apoptotic cell death driven by an activation of the p53 apoptotic signaling pathway. The goal of the present study was to investigate the underlying molecular mechanisms linked to tributyrin-mediated p53 activation. Using in vivo and in vitro models of liver cancer, we demonstrate that an increase in the level of p53 protein in nuclei, a decrease in the level of cytoplasmic p53, and, consequently, an increase in the ratio of nuclear/cytoplasmic p53 in rat preneoplastic livers and in rat and human HCC cell lines caused by tributyrin or sodium butyrate treatments was associated with a marked increase in the level of nuclear chromosome region maintenance 1 (CRM1) protein. Mechanistically, the increase in the level of nuclear p53 protein was associated with a substantially reduced binding interaction between CRM1 and p53. The results demonstrate that the cancer-inhibitory activity of sodium butyrate and its derivatives on liver carcinogenesis may be attributed to retention of p53 and CRM1 proteins in the nucleus, an event that may trigger activation of p53-mediated apoptotic cell death in neoplastic cells.

Interstrain differences in the progression of nonalcoholic steatohepatitis to fibrosis in mice are associated with altered hepatic iron metabolism.

Nonalcoholic fatty liver disease (NAFLD) is a major health problem worldwide. Currently, there is a lack of conclusive information to clarify the molecular events and mechanisms responsible for the progression of NAFLD to fibrosis and cirrhosis and, more importantly, for differences in interindividual disease severity. The aim of this study was to investigate a role of interindividual differences in iron metabolism among inbred mouse strains in the pathogenesis and severity of fibrosis in a model of NAFLD. Feeding male A/J, 129S1/SvImJ and WSB/EiJ mice a choline- and folate-deficient diet caused NAFLD-associated liver injury and iron metabolism abnormalities, especially in WSB/EiJ mice. NAFLD-associated fibrogenesis was correlated with a marked strain- and injury-dependent increase in the expression of iron metabolism genes, especially transferrin receptor (Tfrc), ferritin heavy chain (Fth1), and solute carrier family 40 (iron-regulated transporter), member 1 (Slc40a1, Fpn1) and their related proteins, and pronounced down-regulation of the iron regulatory protein 1 (IRP1), with the magnitude being A/J<129S1/SvImJ

Modulation of intracellular iron metabolism by iron chelation affects chromatin remodeling proteins and corresponding epigenetic modifications in breast cancer cells and increases their sensitivity to chemotherapeutic agents.

Iron plays a vital role in the normal functioning of cells via the regulation of essential cellular metabolic reactions, including several DNA and histone-modifying proteins. The metabolic status of iron and the regulation of epigenetic mechanisms are well-balanced and tightly controlled in normal cells; however, in cancer cells these processes are profoundly disturbed. Cancer-related abnormalities in iron metabolism have been corrected through the use of iron-chelating agents, which cause an inhibition of DNA synthesis, G1-S phase arrest, an inhibition of epithelial-to-mesenchymal transition, and the activation of apoptosis. In the present study, we show that, in addition to these well-studied molecular mechanisms, the treatment of wild-type TP53 MCF-7 and mutant TP53 MDA-MB-231 human breast cancer cells with desferrioxamine (DFO), a model iron chelator, causes significant epigenetic alterations at the global and gene-specific levels. Specifically, DFO treatment decreased the protein levels of the histone H3 lysine 9 demethylase, Jumonji domain-containing protein 2A (JMJD2A), in the MCF-7 and MDA-MB-231 cells and down-regulated the levels of the histone H3 lysine 4 demethylase, lysine-specific demethylase 1 (LSD1), in the MDA-MB-231 cells. These changes were accompanied by alterations in corresponding metabolically sensitive histone marks. Additionally, we demonstrate that DFO treatment activates apoptotic programs in MCF-7 and MDA-MB-231 cancer cells and enhances their sensitivity to the chemotherapeutic agents, doxorubicin and cisplatin; however, the mechanisms underlying this activation differ. The induction of apoptosis in wild-type TP53 MCF-7 cells was p53-dependent, triggered mainly by the down-regulation of the JMJD2A histone demethylase, while in mutant TP53 MDA-MB-231 cells, the activation of the p53-independent apoptotic program was driven predominantly by the epigenetic up-regulation of p21.

Strain-dependent dysregulation of one-carbon metabolism in male mice is associated with choline- and folate-deficient diet-induced liver injury.

Dysregulation of one-carbon metabolism-related metabolic processes is a major contributor to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). It is well established that genetic and gender-specific variations in one-carbon metabolism contribute to the vulnerability to NAFLD in humans. To examine the role of one-carbon metabolism dysregulation in the pathogenesis and individual susceptibility to NAFLD, we used a "population-based" mouse model where male mice from 7 inbred were fed a choline- and folate-deficient (CFD) diet for 12 wk. Strain-dependent down-regulation of several key one-carbon metabolism genes, including methionine adenosyltransferase 1α (Mat1a), cystathionine-ß-synthase (Cbs), methylenetetrahydrofolate reductase (Mthfr), adenosyl-homocysteinase (Ahcy), and methylenetetrahydrofolate dehydrogenase 1 (Mthfd1), was observed. These changes were strongly associated with interstrain variability in liver injury (steatosis, necrosis, inflammation, and activation of fibrogenesis) and hyperhomocysteinemia. Mechanistically, the decreased expression of Mat1a, Ahcy, and Mthfd1 was linked to a reduced level and promoter binding of transcription factor CCAAT/enhancer binding protein ß (CEBPß), which directly regulates their transcription. The strain specificity of diet-induced dysregulation of one-carbon metabolism suggests that interstrain variation in the regulation of one-carbon metabolism may contribute to the differential vulnerability to NFLD and that correcting the imbalance may be considered as preventive and treatment strategies for NAFLD.

Modifying metabolically sensitive histone marks by inhibiting glutamine metabolism affects gene expression and alters cancer cell phenotype.

The interplay of metabolism and epigenetic regulatory mechanisms has become a focal point for a better understanding of cancer development and progression. In this study, we have acquired data supporting previous observations that demonstrate glutamine metabolism affects histone modifications in human breast cancer cell lines. Treatment of non-invasive epithelial (T-47D and MDA-MB-361) and invasive mesenchymal (MDA-MB-231 and Hs-578T) breast cancer cell lines with the glutaminase inhibitor, Compound 968, resulted in cytotoxicity in all cell lines, with the greatest effect being observed in MDA-MB-231 breast cancer cells. Compound 968-treatment induced significant downregulation of 20 critical cancer-related genes, the majority of which are anti-apoptotic and/or promote metastasis, including AKT, BCL2, BCL2L1, CCND1, CDKN3, ERBB2, ETS1, E2F1, JUN, KITLG, MYB, and MYC. Histone H3K4me3, a mark of transcriptional activation, was reduced at the promoters of all but one of these critical cancer genes. The decrease in histone H3K4me3 at global and gene-specific levels correlated with reduced expression of SETD1 and ASH2L, genes encoding the histone H3K4 methyltransferase complex. Further, the expression of other epigenetic regulatory genes, known to be downregulated during apoptosis (e.g., DNMT1, DNMT3B, SETD1 and SIRT1), was also downregulated by Compound 968. These changes in gene expression and histone modifications were accompanied by the activation of apoptosis, and decreased invasiveness and resistance of MDA-MB-231 cells to chemotherapeutic drug doxorubicin. The results of this study provide evidence to a link between cytotoxicity caused by inhibiting glutamine metabolism with alterations of the epigenome of breast cancer cells and suggest that modification of intracellular metabolism may enhance the efficiency of epigenetic therapy.

Histone H3 lysine 9 and H4 lysine 20 trimethylation and the expression of Suv4-20h2 and Suv-39h1 histone methyltransferases in hepatocarcinogenesis induced by methyl deficiency in rats.

The field of cancer epigenetics has received much attention in recent years. However, the relationship of cancer epigenetics with cancer etiology is not clear. Recent studies suggest the involvement of altered DNA methylation and histone modifications in the emergence of epigenetically reprogrammed cells with specific tumor-related phenotypes at premalignant stages of tumor development. In this study, we used a methyl-deficient model of rodent hepatocarcinogenesis to examine the roles of DNA, histone H3 lysine 9 and histone H4 lysine 20 methylation, and the level of the expression of Suv39h1 and Suv4-20h2 histone methyltransferases in the carcinogenic process. We demonstrated that the development of liver tumors was characterized by progressive demethylation of DNA repeats, decrease in histone H4 lysine 20 trimethylation, and a gradual decrease in the expression of Suv4-20h2 histone methyltransferase. A prominent increase in the trimethylation of histone H3 lysine 9 and in the expression of Suv39h1 histone methyltransferase was observed in preneoplastic nodules and liver tumors indicating the promotional role of these epigenetic alterations at later stages of carcinogenesis. The appearance of tumor-specific epigenetic alterations (demethylation of repetitive elements, loss of histone H4 lysine 20 trimethylation, altered expression of Suv4-20h2 and Suv39h1 histone methyltransferases) at preneoplastic stages of hepatocarcinogenesis provides experimental support for the epigenetic hypothesis of tumorigenesis that considers stress-induced epigenetic reprogramming of the cell as an important prerequisite to succeeding mutations.

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 (GSTpi)-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.

Thimerosal neurotoxicity is associated with glutathione depletion: protection with glutathione precursors.

Thimerosol is an antiseptic containing 49.5% ethyl mercury that has been used for years as a preservative in many infant vaccines and in flu vaccines. Environmental methyl mercury has been shown to be highly neurotoxic, especially to the developing brain. Because mercury has a high affinity for thiol (sulfhydryl (-SH)) groups, the thiol-containing antioxidant, glutathione (GSH), provides the major intracellular defense against mercury-induced neurotoxicity. Cultured neuroblastoma cells were found to have lower levels of GSH and increased sensitivity to thimerosol toxicity compared to glioblastoma cells that have higher basal levels of intracellular GSH. Thimerosal-induced cytotoxicity was associated with depletion of intracellular GSH in both cell lines. Pretreatment with 100 microM glutathione ethyl ester or N-acetylcysteine (NAC), but not methionine, resulted in a significant increase in intracellular GSH in both cell types. Further, pretreatment of the cells with glutathione ethyl ester or NAC prevented cytotoxicity with exposure to 15 microM Thimerosal. Although Thimerosal has been recently removed from most children's vaccines, it is still present in flu vaccines given to pregnant women, the elderly, and to children in developing countries. The potential protective effect of GSH or NAC against mercury toxicity warrants further research as possible adjunct therapy to individuals still receiving Thimerosal-containing vaccinations.

Genomic hypomethylation is specific for preneoplastic liver in folate/methyl deficient rats and does not occur in non-target tissues.

Chronic dietary insufficiency of the lipotropic nutrients choline and methionine is hepatocarcinogenic in male rats and certain mouse strains. Despite the fact that DNA hypomethylation is a hallmark of most cancer genomes, the tissue-specific consequences of this alternation with respect to tumorigenesis remain to be determined. In the present study, the folate/methyl deficient model of multistage hepatocarcinogenesis was used to evaluate in vivo alterations in DNA methylation in the liver, the carcinogenesis target tissue, and in non-target tissues, including pancreas, spleen, kidney, and thymus, of male F344 rats. By utilizing the HpaII/MspI-based cytosine extension assay, we demonstrated that the percent of CpG sites that lost methyl groups on both strands progressively increased in liver tissue after 9, 18, and 36 weeks of folate/methyl deficiency. The endogenous activity of DNA methyltransferase in liver of rats fed with folate/methyl deficient diet for the 36-week period gradually increased with time. In contrast, non-target tissues displayed no changes in DNA methylation level or activity of DNA methyltransferase. The failure of DNA methyltransferase to restore and maintain DNA methylation patterns in preneoplastic liver tissue may lead to the establishment of tumor-specific DNA methylation and DNA methyltransferase profiles that are not expressed in normal liver. These results provide additional information about alterations in DNA methylation during early preneoplastic stages of carcinogenesis. They also demonstrate that DNA hypomethylation is localized to tissue that undergoes carcinogenesis, and is not altered in non-target tissues.

Mechanisms of DNA damage, DNA hypomethylation, and tumor progression in the folate/methyl-deficient rat model of hepatocarcinogenesis.

Using the folate/methyl-deficient rat model of hepatocarcinogenesis, we obtained evidence that may provide new insights into a major unresolved paradox in DNA methylation and cancer research: the mechanistic basis for genome-wide hypomethylation despite an increase in DNA methyltransferase activity and gene-specific regional hypermethylation. Previous studies revealed that the methyltransferase binds with higher affinity to DNA strand breaks, gaps, abasic sites, and uracil than it does to its cognate hemimethylated CpG sites, consistent with its ancestral function as a DNA repair enzyme. These same DNA lesions are an early occurrence in models of folate and methyl deficiency and are often present in human preneoplastic cells. We hypothesized that the high-affinity binding of the maintenance DNA methyltransferase to unrepaired lesions in DNA could sequester available enzyme away from the replication fork and promote passive replication-dependent demethylation. In support of this possibility, we found that lesion-containing DNA is less efficiently methylated than lesion-free DNA from folate/methyl-deficient rats and that an increase in DNA strand breaks precedes DNA hypomethylation. Despite an adaptive increase in DNA methyltransferase activity, hemimethylated DNA from folate/methyl-deficient rats is progressively replaced by double-stranded unmethylated DNA that is resistant to remethylation with dietary methyl repletion. In promoter regions, the inappropriate binding of the DNA methyltransferase to unrepaired lesions or mispairs may promote local histone deacetylation, methylation, and regional hypermethylation associated with tumor suppressor gene silencing. These insights in an experimental model are consistent with the possibility that DNA lesions may be a necessary prerequisite for the disruption of normal DNA methylation patterns in preneoplastic and neoplastic cells.

Elevation in S-adenosylhomocysteine and DNA hypomethylation: potential epigenetic mechanism for homocysteine-related pathology.

Chronic nutritional deficiencies in folate, choline, methionine, vitamin B-6 and/or vitamin B-12 can perturb the complex regulatory network that maintains normal one-carbon metabolism and homocysteine homeostasis. Genetic polymorphisms in these pathways can act synergistically with nutritional deficiencies to accelerate metabolic pathology associated with occlusive heart disease, birth defects and dementia. A major unanswered question is whether homocysteine is causally involved in disease pathogenesis or whether homocysteinemia is simply a passive and indirect indicator of a more complex mechanism. S-Adenosylmethionine and S-adenosylhomocysteine (SAH), as the substrate and product of methyltransferase reactions, are important metabolic indicators of cellular methylation status. Chronic elevation in homocysteine levels results in parallel increases in intracellular SAH and potent product inhibition of DNA methyltransferases. SAH-mediated DNA hypomethylation and associated alterations in gene expression and chromatin structure may provide new hypotheses for pathogenesis of diseases related to homocysteinemia.