S-Adenosylhomocysteine enhances DNA damage through increased ß-amyloid formation and inhibition of the DNA-repair enzyme OGG1b in microglial BV-2 cells.

S-Adenosylhomocysteine (SAH) is a risk factor for neurodegenerative diseases such as Alzheimer's disease, for which ß-Amyliod (Aß) formation is a major risk factor. We recently showed that SAH increases Aß formation in mouse microglial BV2 cells. Here, we show that incubation of BV2 cells with SAH (0-500nM) for 6-24h sequentially increased Aß formation, ROS and DNA damage measured as 8-oxo-deoxyguanosine (8-oxo-dG) levels. Pre-incubation of BV2 cells with 20µM ß-secretase inhibitor IV for 30min followed by incubation with SAH (500nM) markedly decreased Aß formation and 8-oxo-dG levels. Treatment with SAH for 24h concentration-dependently inhibited DNA methyltransferase (DNMT1) activity and inhibited DNMT1 binding to Sp1 site of 8-oxoG-DNA glycosylases I (OGG1) promoter and OGG1 protein and mRNA expression at 24h; the latter effect was attributed to hypomethylation of the OGG1 gene promoter, because pre-incubation of cells with betaine (1.0mM for 30 min) markedly prevented the inhibition of OGG1 protein expression induced by SAH. Overall, we demonstrate that SAH increases DNA damage in BV-2 cells possible by increased Aß formation leading to increased formation of ROS. Furthermore, the DNA damage is enhanced by SAH through inhibition of DNMT1 activity and hypomethylation of OGG1 gene promoter.

Effects of S-adenosylhomocysteine and homocysteine on DNA damage and cell cytotoxicity in murine hepatic and microglia cell lines.

Limited research has been performed on S-adenosylhomocysteine (SAH) or homocysteine (Hcy)-evoked cell damage in hepatic and neuronal cells. In this study, we assessed effects of SAH or Hcy on cell cytotoxicity and DNA damage in hepatic and neuronal cells and attempted to find the underlying mechanism. Cell cytotoxicity and DNA damage were evaluated in murine hepatic cells (BNL CL.2 cell line) and microglia cells (BV-2 cell line) with SAH or Hcy treatment for 48 h. The influences of SAH or Hcy on lipid peroxidation and DNA methylation were also measured in both cell lines. SAH (5-20 microM) or Hcy (1-5 mM) dose dependently inhibited cell cytotoxicity and enhanced DNA damage in both types of cells. Furthermore, SAH treatment markedly increased intracellular SAH levels and DNA hypomethylation, whereas Hcy caused minimal effects on these two parameters at much higher concentrations. Hcy significantly induced lipid peroxidation, but not SAH. The present results show that SAH might cause cellular DNA damage in hepatic and microglia cells by DNA hypomethylation, resulting in irreversible DNA damage and increased cell cytotoxicity. In addition, higher Hcy could induce cellular DNA damage through increased lipid peroxidation and DNA hypomethylation. We suggest that SAH is a better marker of cell damage than Hcy in hepatic and microglia cells.

S-Adenosylhomocysteine promotes the invasion of C6 glioma cells via increased secretion of matrix metalloproteinase-2 in murine microglial BV2 cells.

S-Adenosylhomocysteine (SAH) is a risk factor for many diseases, including tumor progression and neurodegenerative disease. In this study, we examined the hypothesis that SAH may indirectly enhance the invasion of C6 glioma cells by induction of matrix metalloproteinase-2 (MMP-2) secreted from the murine microglia BV2 cells. We obtained conditioned medium (CM) by incubating BV2 cells with SAH (1-50nM) for 24 h. We found that the SAH-containing CM (SAH-BV2-CM) strongly enhanced the invasiveness of C6 glioma cells and that this effect increased with increasing concentrations of SAH in the SAH-BV2-CM. The effect of CM could be attributed to its MMP-2 activity, as a result of increased protein and messenger RNA expression of MMP-2 in BV2 cells induced by SAH. In BV2 cells treated with SAH, the binding abilities of nuclear factor-kappa B (NF-kappaB) and stimulatory protein-1 (Sp1) to the MMP-2 promoter were increased, whereas the level of NF-kappaB inhibitor was decreased. In addition, SAH significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3-kinase/serine/threonine protein kinase (or protein kinase B) (PI3K/Akt) proteins but did not affect that of c-Jun NH2-terminal kinase or p38. Pretreatment of BV2 cells with an inhibitor specific for ERK (U0126) markedly abated the expression of ERK and MMP-2. Furthermore, SAH significantly and dose dependently decreased tissue inhibitor of metalloproteinase-2 (TIMP-2) in BV2 cells. Thus, SAH may induce the invasiveness of C6 glioma cells by decreased TIMP-2 expression and increased MMP-2 expression in BV2 cells. The latter effect is likely mediated through the ERK and PI3K/Akt pathways, with increased binding activities of NF-kappaB and Sp1 to the MMP-2 gene promoter.

Oxidative stress-induced regulation of the methionine metabolic pathway in human lung epithelial-like (A549) cells.

The effects of low, moderate and severe oxidative stress on the steady-state levels of the metabolites involved in the transmethylation/transsulfuration pathway were studied in lung epithelial (A549) cells. When cells were exposed to low (0.1 mM) or moderate (1.0 mM) concentrations of hydrogen peroxide (H(2)O(2)) or tert-butylhydroperoxide (t-butOOH), intracellular levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were significantly decreased, while the SAM/SAH ratio remained the same or elevated. Likewise, extracellular levels of SAM and SAH metabolites remained steady or elevated. Both intracellular and extracellular levels of homocyst(e)ine and cyst(e)ine were decreased. Cell contents of serine, cystathionine and methionine were also decreased. Total intracellular glutathione content was decreased only by moderate t-butOOH exposure. When cells were exposed to high concentrations (10mM) of either of the peroxides, extracellular levels of methionine, cystathionine, and total cyst(e)ine were depleted, mostly due to direct oxidation of sulfur amino acids by peroxides, as indicated by oxidative treatment of culture media alone. Similar to low and moderate oxidative conditions, the levels of SAM, SAH, and sulfur amino acids were decreased, while cell SAM/SAH ratio increased. Paradoxically, under high peroxide exposure, extracellular concentrations of SAM, SAH, and cyst(e)ine were increased, indicating cellular release, despite the severe methionine depletion. Intracellular total glutathione was also decreased. The results indicate that lung epithelial cells release high levels of SAM, probably as an adaptive response to increased oxidative stress, even when the substrate for SAM formation, methionine, is critically depleted.

Synergistic effects of S-adenosylhomocysteine and homocysteine on DNA damage in a murine microglial cell line.

BACKGROUND: Homocysteine (Hcy) and S-adenosylhomocysteine (SAH) are 2 major metabolites of methionine. However, little is known about their interactions in human diseases. METHODS: We determined the interaction of Hcy with SAH on DNA damage (measured as comet formation) and DNA hypomethylation (assayed as 5-methyldeoxycytidine, 5-mdc) in BV-2 cells (immortalized murine microglia). RESULTS: Hcy at 100 micromol/l and SAH at 4 micromol/l alone caused little DNA strand breaks, whereas 100 micromol/l Hcy in combination with 0.5 to 4 micromol/l SAH led to marked DNA damage and uracil misincorporation. The combination of 100 micromol/l Hcy with 4 micromol/l SAH (SAH+Hcy) significantly increased intracellular H(2)O(2), and the DNA damage induced by SAH+Hcy was strongly inhibited by addition of superoxide dismutase, catalase or desferrioxamine, suggesting the involvement of reactive oxygen species. DNA damage induced by SAH+Hcy may also involve DNA hypomethylation (i.e., decreased %5-mdc) because of the high correlation between them. The effects induced by SAH+Hcy were specific to SAH but not to Hcy because they were markedly decreased by replacing SAH with adenosine (4.0 micromol/l) but was not affected by replacing Hcy with cysteine (100 micromol/l). CONCLUSION: SAH in combination with Hcy can cause synergistic DNA damage in BV-2 cells. It remains to be seen whether some of the Hcy-related diseases may be caused by a collaborative action of Hcy with SAH.

S-adenosylhomocysteine, but not homocysteine, is toxic to yeast lacking cystathionine beta-synthase.

Elevated plasma homocysteine is associated with a variety of diseases in humans including coronary heart disease, stroke, peripheral vascular disease, and birth defects. However, the mechanism by which plasma homocysteine affects cells is unknown. We have examined the growth of isogenic wild-type and cystathionine beta-synthase (CBS) deficient yeast in response to homocysteine and its immediate metabolic precursor, S-adenosylhomocysteine (SAH). CBS deficient yeast export significantly more homocysteine into the media than wild-type yeast and have elevated internal pools of homocysteine and SAH. We found that 5 mM homocysteine added to the media had very little effect on the growth of wild-type or CBS deficient yeast, although intracellular homocysteine concentrations increased five- to tenfold. In contrast, as little as 25 microM S-adenosylhomocysteine inhibited the growth of CBS deficient yeast, but had no effect on wild-type yeast. Measurements of the intracellular S-adenosylmethionine (SAM) and SAH indicate that CBS deficient yeast contain reduced SAM/SAH ratios relative to wild-type, and this ratio is further reduced by adding SAH to the media. Growth inhibition by SAH in CBS deficient yeast can be totally reversed by addition of SAM to the media, indicating that the ratio and not absolute level is critical for cell growth. These results suggest that CBS plays a key role in the regulation of the SAM/SAH ratio inside cells and that excessive perturbations of this ratio can inhibit growth. We hypothesize that elevated extracellular homocysteine present in humans may reflect an altered intracellular SAM/SAH ratio and that this may be related to disease pathogenesis.

S-adenosyl-L-homocysteine: a non-cytotoxic hypomethylating agent.

The cytotoxic effect caused by the hypomethylating agent S-adenosyl-L- homocysteine (SAH) was compared with that of two drugs commonly used to induce DNA hypomethylation, 5-azacytidine and 5-aza-2'-deoxycytidine. Two in vitro cytotoxicity tests, the tetrazolium MTT assay and the intracellular lactate dehydrogenase (LDH) activity test, suggest that SAH induces hypomethylation without causing any cytotoxic effect. We propose the use of SAH as a non-cytotoxic agent which may be more suitable for inducing experimental DNA hypomethylation.

S-adenosylhomocysteine toxicity in normal and adenosine kinase-deficient lymphoblasts of human origin.

The human lymphoblast line WI-L2 is subject to growth inhibition by a combination of the adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4.) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and adenosine. Although adenosine-induced pyrimidine starvation appears to contribute to this effect, uridine only partially reverses adenosine toxicity in WI-L2 and not at all in strain 107, an adenosine kinase-(ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) deficient derivative of WI-L2. Treatment of both cell lines with EHNA and adenosine leads to striking elevations in intracellular S-adenosyl-L-homocysteine (AdoHcy), a potent inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methylation reactions. The methylation in vivo of both DNA and RNA is inhibited by concentrations of EHNA and adenosine that elevate intracellular AdoHcy. Addition of 100 muM L-homocysteine thiolactone to cells treated with EHNA and adenosine enhances adenosine toxicity and further elevates AdoHcy to levels approximately 60-fold higher than those obtained in the absence of this amino acid, presumably by combining with adenosine to form AdoHcy in a reaction catalyzed by S-adenosylhomocysteine hydrolase (EC 3.3.1.1). In the adenosine kinase-deficient strain 107, a combination of ADA inhibition and L-homocysteine thiolactone markedly increases intracellular AdoHcy and inhibits growth even in the absence of exogenous adenosine. These results demonstrate a form of toxicity from endogenously produced adenosine and support the view that AdoHcy, by inhibiting methylation, is a mediator of uridine-resistant adenosine toxicity in these human lymphoblast lines. Furthermore, they suggest that AdoHcy may play a role in the pathogenesis of the severe combined immunodeficiency disease found in most children with heritable ADA deficiency.