M.S. Yang

The ginsenoside protopanaxatriol protects endothelial cells from hydrogen peroxide-induced cell injury and cell death by modulating intracellular redox status

Ginsenosides, the active components of the famous Chinese herb ginseng, have been suggested to possess cardiovascular-protective effects. The mechanism of ginsenosides is believed to be associated with their ability to prevent cellular oxidative stress. The purpose of this study was to explore the cytoprotective effects of the ginsenoside protopanaxatriol (PPT) on hydrogen peroxide (H(2)O(2))-induced endothelial cell injury and cell death. Pretreatment of human umbilical vein endothelial cells (HUVECs) with PPT for 24 h was able to protect the cells against H(2)O(2)-induced injury. In addition to cell death, pretreatment with PPT could also reduce H(2)O(2)-induced DNA damage, overactivation of the DNA repair enzyme PARP-1, and concomitant depletion of the intracellular substrate NAD(+). Furthermore, PPT could reverse the decrease in ATP/ADP ratio caused by H(2)O(2). The metabolism of glutathione was also changed. H(2)O(2) could induce a significant decrease in GSH level resulting in a decrease in the GSH/GSSG ratio. This could be prevented by pretreatment with PPT. The action was associated with increasing activities of the GSH-metabolizing enzymes glutathione reductase and glutathione peroxidase. These findings suggest that the ginsenoside PPT could protect HUVECs against H(2)O(2)-induced cell death via its action against oxidative stress, which may be responsible for the cardiovascular-protective action of ginseng.

MAPK regulate p53-dependent cell death induced by benzo[a]pyrene: involvement of p53 phosphorylation and acetylation.

Benzo[a]pyrene (BaP) is a potentially genotoxic and cytotoxic environmental pollutant. Previous studies showed that exposure of HepG(2) cells to BaP causes necrotic cell death [Lin, T., Yang, M.S., 2007b. Cell death induced by benzo[a]pyrene in the HepG(2) cells is dependent on PARP-1 activation and NAD depletion. Toxicology 245, 147-153]. In the present study, the signaling pathways associated with this response was studied. BaP induced accumulation and activation of p53 in HepG(2) cells, which occurred as early as 12h after exposure. Activation of p53 was evidenced by its phosphorylation at serine 15 (Ser15) and acetylation at lysine 382 (Lys382). Chemical inhibition and siRNA-mediated knockdown of p53 expression suppressed its phosphorylation as well as cell death. BaP also activated p38 MAPK and ERK, but not JNK, at 6h after exposure. SB203580 and PD98059, specific inhibitors of p38 MAPK and ERK, respectively, suppressed phosphorylation of p53 at Ser15, but the accumulation of p53 was only moderately reduced. Acetylation of p53 at Lys 382 was not affected by these inhibitors, suggesting that acetylation stabilizes p53 in response to DNA damage. SB203580 and PD98059 prevented downstream energy failure and BaP-induced cell death. Similar results were obtained with siRNA against two isoforms of p38 MAPK, p38alpha and p38beta. Wortmannin, selective inhibitor of DNA-PK and ATM/ATR, abolished p53 phosphorylation, indicating an involvement of multiple pathways of p53 phosphorylation upon exposure to BaP. In summary, the current study demonstrated that both MAPK and p53 activation are required for BaP-induced necrotic cell death. The results also provide a novel model for studying the regulation between p53 and p38 MAPK in the progression of cellular necrosis.

Chemical and ecotoxicological analyses of sediments and elutriates of contaminated rivers due to e-waste recycling activities using a diverse battery of bioassays.

A multi-trophic, multi-exposure phase assessment approach was applied to characterize the toxicity of sediments collected from two rivers in Guiyu, China, an e-waste recycling centre. Elutriate toxicity tests (bacterium Vibrio fischeri and microalga Selenastrum capricornutum) and whole sediment toxicity test (crustacean Heterocypris incongruens) showed that most sediments exhibited acute toxicity, due to elevated heavy metals and PAHs levels, and low pH caused by uncontrolled acid discharge. The survival rates of crustaceans were negatively (p < 0.05) correlated with total PAHs in sediments (411-1755 mg kg(-1)); EC50s of V. fischeri on the elutriates were significantly correlated with elutriate pH (p < 0.01). Significant (p < 0.05) correlations between the induction of hepatic metallothionein in tilapia (Oreochromis mossambicus) and metal concentrations (Cu, Zn, Pb) in sediments were also observed, when fish were fed with diets containing sediment. The results showed that uncontrolled e-waste recycling activities may bring adverse effects to local aquatic ecosystem.

Benzo[a]pyrene-induced necrosis in the HepG2 cells via PARP-1 activation and NAD+ depletion

Benzo[a]pyrene (BaP), a member of polycyclic aromatic hydrocarbons (PAH), has been reported to induce cell death in various cell types. However, the underlying mechanisms are controversial. In the present study, we report that BaP induces necrotic cell death in human hepatoma (HepG(2)) cells. The process is dependent on the activation of poly(ADP-ribose)polymerase-1 (PARP-1), a nuclear enzyme responsible for repairing DNA damage. Once activated, PARP-1 catalyzes the formation of ADP-ribose polymers on acceptor proteins at the expense of NAD(+). Incubation of cells with high extracellular concentration of NAD(+) (5mM) after BaP treatment caused an elevation in intracellular NAD(+) level and blocked cell death. Inhibitor of PARP-1 suppressed both overactivation of PARP-1 activity and NAD(+) depletion. Moreover, addition of pyruvate (5mM), but not glutamate (5mM) or glutamine (5mM), could restore ATP production and prevent cell death. These results elucidated a sequence of events linking cellular metabolism to the progression of cell death induced by this organic toxicant.

Capillary liquid chromatographic-high-resolution mass spectrometric analysis of ribonucleotides.

A method of capillary HPLC-high-resolution MS was developed for the trace analysis of ATP, GTP, dATP and dGTP. Dimetylhexylamine (DMHA) was used as ion-pairing agent for the HPLC retention and separation of the nucleotides and positive ion electrospray time-of-flight MS was used for the detection. The application of capillary HPLC allowed minimal usage of DMHA while providing excellent peak retention and resolution, which significantly reduced the ion suppression in electrospray ionization-MS analysis and thus increased the sensitivity. Adduct ions of nucleotides and DMHA were used as quantitative ions in order to achieve the best sensitivity. DMHA concentration at 5 mM in the aqueous mobile phase at pH 7 found to be the optimal conditions for the C18 capillary column. The method was applied to determine ATP level in cultured C6 glioma cells that were treated with toxic concentrations of Zn. The results showed that the cellular ATP level decreased from 2.7 pmol/cell (<10% cell death) in average control cell samples to 0.36 pmol/cell as the concentration of Zn increased to 120 mg/l (>35% cell death) in culture medium.

Cytoprotective Effect of 20(S)-Rg3 on Benzo[a]pyrene-Induced DNA Damage

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon ubiquitously existing in the environment. Its metabolites have been shown to cause DNA damage and cellular dysfunction in humans. Panax ginseng C.A. Meyer is a Chinese medicinal herb, and ginsenosides are the main active constituent of ginseng. Accumulating evidence had indicated that ginseng extract and ginsenosides possess cytoprotective effects. In this study, the protective effect of ginsenosides on BaP-induced DNA damage in human dermal fibroblasts (HDFs) and HepG2 cells was investigated. The genotoxic effect of BaP was measured by the comet assay. Results showed that tail moment was increased in BaP-treated cells, but cotreatment of ginsenoside 20(S)-Rg3 can significantly decrease BaP-induced DNA damage. A downstream mechanistic study revealed that 20(S)-Rg3 increased the gene expression of an important phase II detoxifying enzyme NAD(P)H:quinine oxidoreductase 1. The effect was also associated with the activation of protein kinase B (Akt) and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). These results indicated that 20(S)-Rg3 might protect HDFs from BaP-induced DNA damage through the activation of the phosphatidylinositol 3-kinase/Akt/Nrf2 pathway. Our results also demonstrated that 20(S)-Rg3 is a functional ligand of pregnane X receptor (PXR), a nuclear receptor that mediates the induction of drug clearance pathways. Subsequent knockdown of PXR expression by small interfering RNA confirmed the involvement of PXR on the protective effects of 20(S)-Rg3 against BaP-induced DNA damage. In summary, ginsenoside 20(S)-Rg3 can protect against BaP-induced genotoxicity in human cells, suggesting that ginseng may serve as a natural cytoprotective agent against environmental carcinogens.

Increase in intracellular free/bound NAD[P]H as a cause of Cd-induced oxidative stress in the HepG2 cells

The present study shows the use of confocal autofluorescence spectroscopy coupled with the time-resolved fluorescence decay analysis to measure changes in FAD/NAD[P]H and free/bound NAD[P]H in HepG(2) cells at 0.5, 1.5, 3 and 4.5h after exposure to cadmium chloride (Cd). These changes were compared to changes in GSSG/GSH and production of reactive oxygen radicals (ROS) production. The results demonstrated that both FAD/NAD[P]H and GSSG/GSH increased significantly upon exposure to Cd. The change in GSSG/GSH occurred as early as 1.5h after treatment while the change in FAD/NAD[P]H did not occur until 3h after exposure. Production of ROS was also increased at 1.5h. The ratio of free/bound NAD[P]H was studied. It was demonstrated that free/bound NAD[P]H increased significantly as early as 0.5h and remained elevated until 4.5h after treatment with Cd. The present study provides novel data to show that changes in NAD[P]H metabolism precedes the increase in ROS production and cellular oxidative stress (increase GSSG/GSH, FAD/NAD[P]H). It is suggested that Cd causes a release of NAD[P]H, an important cofactor for electron transfer, from its normal protein binding sites. This may result in a disruption of the activity of the enzyme and proteins, and may lead to the subsequent toxic events.

Changes in endogenous Zn and Cu distribution in different cytosolic protein fractions in mouse liver after administration of a single sublethal dose of CdCl2

The time course of change in tissue Cd, Cu and Zn contents, their distribution in cellular protein fractions as well as the profile of MT gene expression in mouse liver was described over a 7 days period following a single intraperitoneal injection of 2 mg/kg of CdCl(2). The result showed that Cd accumulated rapidly in mouse liver. Between 1 h and 7 days after administration, over 18% of the total Cd administered were found in the liver. Cd administration was also associated with the overexpression of the MT-mRNA. However, the time course of induction was not parallel to the change in tissue Cd content. When separated on a Sephadex G-75 column, majority of Cd was found to bind to the fractions known to contain the metal-binding protein, metallothionein (MT). From day 2 after Cd administration, a small amount of the metal was also found associated with the high molecular weight (HMW) proteins. In addition to Cd, tissue Zn content was affected most during the entire study. There was a significant decrease in tissue Zn content during the initial 8 h but tissue Zn content increased significantly throughout the following 6 days. At 1-7 days, majority of Zn was associated with the HMW protein fraction. Although there was no significant change in total tissue Cu content, distribution of Cu in different protein fractions was detected. While in control animals, Cu was mainly associated with the HMW proteins, some was found in the MT fraction on the second day. On the 7th day, Cu distribution had deteriorated. Together with changes seen in Cd, the results might suggest that injury had occurred in the tissue at this time. The results of the present study showed that Cd caused a change in subcellular distribution of tissue endogenous metals, which might reflect alteration of cellular functional activities.

Effects of ginsenosides Re and Rg3 on intracellular redox state and cell proliferation in C6 glioma cells

BackgroundCellular redox state is important to cell growth and death. The growth of tumor cells may be modulated by intracellular reduced glutathione/oxidized glutathione (GSH/GSSG). The present study aims to investigate the effects of ginsenosides Re and Rg3 on cellular redox state and cell proliferation in C6 glioma cells.MethodsCultured C6 glioma cells were exposed to various concentrations of either Rg3 or Re for 24 hours. Cell growth and death were measured by the BrdU incorporation assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay respectively. Cellular redox state was determined by free radical production using flow cytometry and GSH/GSSG using spectrofluorometry.ResultsAt a sub-lethal concentration, Re suppressed cell proliferation with a significant decrease in BrdU incorporation. Re did not increase reactive oxygen species (ROS) production but increased GSH/GSSG via increased activity of gamma glutamylcystenyl synthase (γ-GCS). In contrast, Rg3 increased free radical production and reduced GSH/GSSG. The effects of Rg3 were probably due to increased activity of glutathione peroxidase (GPx).ConclusionRe and Rg3 alter cellular redox state of C6 glioma cells in opposite directions. Changes in cellular redox state induced by Re and Rg3 are correlated with the proliferation rates of C6 glioma cells.

Analysis of multiple metabolomic subsets in vitro: methodological considerations.

Abstract Metabolomic analysis is a technology which seeks to provide a comprehensive profile of all the metabolites present in a biological sample (; ; ). Metabolomics is not a new science, but emerging as an exciting application which can span the scope of biotechnology and medicine, providing metabolic profile and a complement to the genomic and proteomic data (). Studies have already begun to explore the effects of toxicological, pharmaceutical, nutritional, and environmental intervention and to build integrated databases of metabolite concentrations throughout biological systems, ranging from microbes to plants to human and research animal populations (; ; ; ). This may provide a tool for discovering a novel pathway, or determining the relationship of these metabolite concentrations to disease, and the extent to which nutrition can modulate metabolite concentrations (). Metabolites are the products of enzymatic processes. Their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes (). While the amount of an enzyme protein in a biological system could be quantified by using specific antibodies or by measuring the mRNA responsible for their production, factors such as the availability of cofactors and coenzymes, feedback regulation, pH, compartmentalization, etc. could affect their activity, thus, in turn, affect the metabolites (i.e., substrates and their enzymatic products) levels. There have been a number of different interpretations of “metabolites.” In pharmacological and toxicological studies, metabolites could be considered as the enzymatic degradative products of drugs (; ; ; ; ). In other studies, metabolites could be referred to as amino acids (), keto acids (), or lipids (), which are substrates and products of selected metabolic pathways. proposed using “subset” to indicate the type of metabolites to be studied. Thus, the amino acid subset, for example, would indicate the levels of all the amino acids in a sample and lipomic subset would indicate the levels of all metabolites involved in lipid metabolism. In biochemical, physiological, and toxicological studies, animal models are preferred. In fact, a large amount of information on metabolites balance has been accumulated for a number of pharmacological, toxicological, and pathological conditions (; ; ; ; ; ; ; ; ,b; Gupta and Dettbarn 2003). Only recently has attention been directed towards studying metabolites in cell culture. Cell culture, with intact membrane and cytoplasmic organelles that operate under well understood biochemical and molecular events, provides a simple model that is best used for studying the basic mechanisms of toxicological and pharmacological actions of xenobiotics. Other advantages of using cell culture are to lighten the heavy workload and complications involved in performing animal studies, as well as to reduce the use of animals, which is also advocated on the ground of good animal ethics. To correlate the results in vivo, the data need to be transformed taking into consideration the level of toxicant at the site of action.