Modulation of the S-Adenosylmethionine to S-Adensoyhomocyseine Ratio: Effect on Transmethylation

Regulation of transmethylation by lowering the molar ratio of S-adenosylmethionine to S-sdenosylhomocysteine (SAM: SAH ration) was studied. Reduction of the ration in vitro caused a decrease in the activities of rat liver thiopurine methyltransferase (TPMT), thiol methyl-transferase (TMT), and protien(lysine)methyltransferase (PLMT). Furthermore, rats injected with D,L-homocysteine thiolactone had a reduction in the hepatic SAM:SAH ration, as well as a decrease in the activities of TPMT and PLMT, but not TMT, in liver. Reduction of the hepatic SAM:SAH ratio in rats fed a pyridoxine-deficient diet resulted in a decrease in the activity of PLMT, but not TPMT and TMT, in liver. Moreover, the levels of total acid-soluble carnitine (ASCNE) were lowered in skeletal muscle and heart of these rats, but palmitate oxidation in vitro was not impared in either tissue. On the other hand, the rate of palmitate oxidation in skeletal muscle decreased in rats subjected to an apparent feed restriction, despite normal levels of tissue ASCNE and elevated cytochrome oxidase activity. Phospholipid methylation was impaired during pyridoxine deficiency, above and beyond that seen during feed restriction. A reduction in the hepatic microsomal level of phosphatidylcholine and an elevation of phosphatidylethanolamine was found, incongruous with an increase in the activity of phosphatidylethanolamine metyltransferase. Pyridoxine-deficient rats displayed no significant change in the activity of guanidoacetate methyltransferase in liver, but a decrease in arginine-glycine transamidinase activity of the kidney. The concentrations of creatine in liver and skeletal muscle of these animals increased, once again above and beyond that observed in feed-restricted rats alone. No change in urinary creatinine excretion was demonstrated. The data indicate that under certain conditions, the SAM:SAH ratio may regulate some biochemical processes related to transmethylation. Cellular compartmentalization and nutritional state may perhaps determine if control is possible in vivo

Fluid leak indicator

A fluid leak indicator (30) for detecting and indicating leaks in visually inaccessible fluid tubing joints (20, 21), such as those obstructed by insulation (24), includes a bag system (25) and a wicking system (30) surrounding or wrapping the joints (20, 21) under the visual obstructing material (24). Leaking fluid is collected in the bag (25) or on the wicking material (34) where it is conducted along the wicking material (34) to a visibly accessible capturing transparent indicator bulb (35) for providing a visual indication of the leak without requiring a chemical change in the capturing indicator bulb (35)

Redox-sensitive mechanisms of phytochemical-mediated inhibition of cancer cell proliferation (Review)

Phytochemicals are potential cancer chemopreventive agents, based partly on cellular research establishing that phytochemicals inhibit the proliferation of cancer cells. To elucidate the mechanism of phytochemicals, a basic understanding is needed of what stimulates cancer cell proliferation. Cancer cells, particularly those that are highly invasive or metastatic, may require a certain level of oxidative stress to maintain a balance between undergoing either proliferation or apoptosis. They constitutively generate large but tolerable amounts of H2O2 that apparently function as signaling molecules in the mitogen-activated protein kinase pathway to constantly activate redox-sensitive transcription factors and responsive genes that are involved in the survival of cancer cells as well as their proliferation. With such a reliance of cancer cells on H2O2, it follows that if the excess H2O2 can be scavenged by phenolic phytochemicals having antioxidant activity, the oxidative stress-responsive genes can be suppressed and consequently cancer cell proliferation inhibited. On the other hand, phenolic phytochemicals and another group of phytochemicals known as isothiocyanates can induce the formation of H2O2 to achieve an intolerable level of high oxidative stress in cancer cells. As an early response, the stress genes are activated. However, when the critical threshold for cancer cells to cope with the induced oxidative stress has been reached, key cellular components such as DNA are damaged irreparably. In conjunction, genes involved in initiating cell cycle arrest and/or apoptosis are activated. Therefore, phytochemicals can either scavenge the constitutive H2O2 or paradoxically generate additional amounts of H2O2 to inhibit the proliferation of cancer cells

Increased GADD Gene Expression in Human Colon Epithelial Cells Exposed to Deoxycholate

The colonic epithelium is often exposed to high concentrations of secondary bile acids, which stresses the epithelial cells, leading potentially to activation of stress- response genes. To examine this possibility in vitro, the purpose of this study was to determine if expression of certain growth arrest and DNA damage- inducible genes (GADD) is upregulated in human colonic epithelial cells exposed to deoxycholate (DOC). DNA macroarray screening of a small cluster of stress/apoptosis-related genes in DOC-treated HCT-116 colonocytes revealed clearly higher expression of only GADD45, which was confirmed by gene-specific relative RT-PCR analysis. Subsequently, it was found that DOC also increased GADD34 mRNA expression. However, mRNA expression of GADD153 was increased most markedly in DOC-treated HCT-116 colonocytes, which express wild-type p53. However, the upregulation of GADD34, GADD45, and GADD153 mRNA expression apparently did not require p53, based on the finding that DOC increased expression of all three GADD genes in HCT-15 colonocytes, which express mutant p53. In further studying GADD153 in particular, the effect of DOC on GADD153 mRNA was prevented by actinomycin-D (Act-D), but not by antioxidants or MAPK inhibitors. DOC also caused GADD153 protein to be expressed in close parallel with increased GADD153 mRNA expression. Induction of GADD153 protein by DOC was prevented by either anisomycin or cycloheximide. These findings suggest that DOC-induced upregulation of GADD153 mRNA expression occurred at the level of transcription without involving reactive oxygen species and MAPK signaling, and that the expression of GADD153 protein was due also to translation of pre-existing, and not just newly synthesized, mRNA

Effect of copper deficiency on oxidative DNA damage in Jurkat T-lymphocytes

The micronutrient copper is a catalytic cofactor for copper, zinc superoxide dismutase and ceruloplasmin, which are two important antioxidant enzymes. As such, a lack of copper may promote oxidative stress and damage. The purpose of this study was to determine the effect of copper deficiency on oxidative damage to DNA in Jurkat T-lymphocytes. To induce copper deficiency, cells were incubated for 48 h with 5-20 µM 2,3,2-tetraamine (2,3,2-tet), a high affinity copper chelator. Such treatment did not affect cell proliferation/viability, as assessed by measuring mitochondrial reduction of WST-1 reagent (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate). Furthermore, the induction of copper deficiency did not promote oxidative DNA damage as evaluated by the comet assay. Comet scores were 15 ± 0 and 16 ± 1 for control and copper-deficient cells, respectively. However, the copper-deficient cells sustained greater oxidative DNA damage than the control cells (comet scores of 175 ± 15 and 50 ± 10, respectively) when both were oxidatively challenged with 50 µM hydrogen peroxide (H2O2), Supplemental copper but not zinc or iron prevented the potentiation of the H2O2-induced oxidative DNA damage caused by 2,3,2-tet. These data suggest that copper deficiency compromises the antioxidant defense system of cells, thereby increasing their susceptibility to oxidative DNA damage

Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA

Phenolic phytochemicals are thought to promote optimal health, partly via their antioxidant effects in protecting cellular components against free radicals. The aims of this study were to assess the free radical-scavenging activities of several common phenolic phytochemicals, and then, the effects of the most potent phenolic phytochemicals on oxidative damage to DNA in cultured cells. Epigallocatechin gallate (EGCG) scavenged the stable free radical, a,a-diphenyl-ß-picrylhydrazyl (DPPH), most effectively, while quercetin was about half as effective. Genistein, daidzein, hesperetin, and naringenin did not scavenge DPPH appreciably. Jurkat T-lymphocytes that were pre-incubated with relatively low concentrations of either EGCG or quercetin were less susceptible to DNA damage induced by either a reactive oxygen species or a reactive nitrogen species, as evaluated by the comet assay. More specifically, control cells had a comet score of only 17 ± 5, indicating minimal DNA damage. Cells challenged with 25 pM hydrogen peroxide (H2O2) or 100 µM 3-morpholinosydnonimine (SIN-1, a peroxynitrite generator) had comet scores of 188 ± 6 and 125 ± 12, respectively, indicating extensive DNA damage. The H2O2-induced DNA damage was inhibited with 10 µM of either EGCG (comet score: 113 ± 23) or quercetin (comet score: 82 ± 7), Similarly, the SIN-1-mediated DNA damage was inhibited with 10 µM of either EGCG (comet score: 79 ± 13) or quercetin (comet score: 72 ± 17), In contrast, noticeable DNA damage was induced in Jurkat T-lymphocytes by incubating with 10-fold higher concentrations (i.e., 100 pM) of either EGCG (comet score: 56 ± 17) or quercetin (comet score: 64 ± 13) by themselves. Collectively, these data suggest that low concentrations of EGCG and quercetin scavenged free radicals, thereby inhibiting oxidative damage to cellular DNA. But, high concentrations of either EGCG or quercetin alone induced cellular DNA damage

Curcumin-induced GADD153 gene up-regulation in human colon cancer cells

Ingestion of plant products containing the phenolic phytochemical, curcumin, has been linked to lower incidences of colon cancer, suggesting that curcumin has cancer chemopreventive effects. Supporting this suggestion at the cellular level, apoptosis occurs in human colon cancer cells exposed to curcumin. However, the mechanism is unclear, prompting this investigation to further clarify the molecular effects of curcumin. HCT-116 colonocytes were incubated with 0-20 mM curcumin for 0-48 h. In concentration-dependent and time-dependent manners, curcumin induced DNA damage, resulting later in the appearance of cellular features characteristic of apoptosis. To identify a potential pro-apoptotic gene that could be responsive to the DNA damage in curcumin-treated cells, growth arrest and DNA damage-inducible gene 153 (GADD153) was considered. Curcumin increased GADD153 mRNA (and also protein) expression, which was prevented by actinomycin D and also by a broad protein kinase C inhibitor, but not by selective MAPK inhibitors. These findings suggest that curcumin-induced up-regulation of GADD153 mRNA expression was at the level of transcription, but apparently without depending on upstream MAPK. In determining the involvement of reactive oxygen species in mediating the effect of curcumin on GADD153, the antioxidants pyrrolidine dithiocarbamate and N-acetylcysteine (NAC), but neither a-tocopherol nor catalase, also blunted or prevented up-regulation of GADD153 mRNA expression caused by curcumin. Most noteworthy, when NAC was tested, it inhibited the DNA damage and apoptosis caused by curcumin. Because expression of GADD153 protein was detected before the appearance of apoptotic features, this observation raises the possibility that GADD153 protein might be important for curcumin-induced apoptosis

Upregulation of haeme oxygenase-1 by zinc in HCT-116 cells

Haeme oxygenase-1 (HO-1) is often viewed as a cytoprotective gene. Toxic heavy metals induce HO-1, but it is unclear whether particular metal micronutrients also induce HO-1. Hence, the ability of exogenously-added copper, iron and zinc to influence HO-1 expression in HCT-116 cells was evaluated. Under the chosen experimental conditions, only zinc noticeably increased the expression of HO-1 mRNA and protein. Concurrently, zinc decreased non-protein thiol levels to a certain extent, but zinc did not increase the production of reactive oxygen species (ROS). Moreover, ascorbate and Trolox did not inhibit zinc-induced HO-1 upregulation. In contrast, deferoxamine blunted the induction of HO-1 mRNA, protein, and enzymatic activity caused by zinc. Additionally, N-acetylcysteine and Tiron inhibited zinc-induced HO-1 upregulation and also nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Collectively, these findings suggest that zinc at above normal levels upregulates HO-1 expression in HCT-116 cells in a ROS-independent manner

Protection of human colon epithelial cells against deoxycholate by rottlerin

The bile salt, deoxycholate (DOC), can harm cells and cause disease. Hence, there is interest in identifying compounds capable of protecting cells against DOC. In HCT-116 colon epithelial cells, DOC increased generation of reactive oxygen species and caused DNA damage and apoptosis. These effects of DOC were inhibited by rottlerin, which is a phenolic compound of plant origin. In elucidating its mechansim, rottlerin prevented the release of cytochrome c from mitochondria into cytosol, and also prevented the cleavage of caspase-3. Yet, rottlerin by itself markedly decreased mitochondrial membrane potential and increased mitochondrial superoxide production, but this did not result in cytochrome c release or in caspase-3 cleavage. At a higher test concentration, two other phenolic phytochemicals, namely, quercetin and resveratrol, were each able to largely prevent the occurrence of apoptosis in cells exposed to DOC. In contrast, epigallocatechin gallate, curcumin, and genistein were ineffective

Epigallocatechin Gallate Protects U937 Cells Against Nitric Oxide-induced Cell Cycle Arrest and Apoptosis

Ingesting phenolic phytochemicals in many plant products may promote health, but the effects of phenolic phytochemicals at the cellular level have not been fully examined. Thus, it was determined if the tea phenolic phytochemical, epigallocatechin gallate (EGCG), protects U937 human pro-monocytic cells against the nitrogen free radical, nitric oxide (•NO). Cells were incubated for 4-6 h with 500 µM S-nitrosoglutathione (GSNO), which generates •NO, but this did not induce single-strand breaks in DNA. Nevertheless, 82 ± 4% of GSNO-treated cells, compared to only 39 ± 1% of untreated cells, were arrested in the G1-phase of the cell cycle. However, dosing the GSNO-treated cells with 9, 14, or 18 µg/ml of EGCG resulted in only 74 ± 8%, 66 ± 1%, and 43 ± 3% of the cells, respectively, in the G1-phase. Exposing cells to GSNO also resulted in the emergence of a sub-G1 apoptotic cell population numbering 14 ± 3%, but only 5 ± 2%, 5 ± 1%, and 2 ± 0% upon dosing of the GSNO-treated cells with 9, 14, and 18 µg/ml of EGCG, respectively. Furthermore, exposing cells to GSNO resulted in greater cell surface binding of annexin V-FITC, but binding was 41-89% lower in GSNO-treated cells dosed with EGCG. Collectively, these data suggest that •NO or downstream products induced cell cycle arrest and apoptosis that was not due to single-strand breaks in DNA, and that EGCG scavenged cytotoxic •NO or downstream products, thus reducing the number of cells in a state of cell cycle arrest or apoptosis