Rescue from neonatal death in the murine model of hereditary tyrosinemia by glutathione monoethylester and vitamin C treatment

Hereditary tyrosinemia type 1 (HT1) is a recessive disease caused by a deficiency of the enzyme fumarylacetoacetate hydrolase (FAH) that catalyzes the conversion of fumarylacetoacetate (FAA) into fumarate and acetoacetate. In mice models of HT1, FAH deficiency causes death within the first 24h after birth. Administration of 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3 cyclohexanedione (NTBC) prevents neonatal death in HT1 mice, ameliorates the HT1 phenotype but does not prevent development of hepatocellular carcinoma later on. FAA has been shown to deplete cells of glutathione by forming adducts. We tested whether a combination of a cell membrane permeable derivative of glutathione, glutathione monoethylester (GSH-MEE) and vitamin C could provide an alternative effective treatment for HT1. GSH-MEE (10 mmol/kg/j)/vitamin C (0.5 mmol/kg/j) treatment was given orally to pregnant/nursing female mice. While FAH-/- pups died in absence of treatment, all FAH-/- pups survived the critical first 24h of life when the mothers were on the GSH-MEE/vitamin C treatment and showed normal growth until postnatal day 10 (P10). However, after P10, pups showed failure to thrive, lethargy and died around P17. Thus, GSH-MEE/vitamin C supplementation could rescue the mice model of HT1 from neonatal death but it did not prevent the appearance of a HT1 phenotype in the second week after birth.     

In vitro and in vivo neuroprotection by gamma-glutamylcysteine ethyl ester against MPTP: relevance to the role of glutathione in Parkinson's disease

Glutathione is an abundant intracellular thiol antioxidant whose levels are reduced both in Parkinson's disease itself and in a widely used animal model of the disorder, systemic MPTP administration. Previous in vitro work from our laboratory has suggested that glutathione depletion may be directly responsible for mitochondrial dysfunction, which ultimately leads to dopaminergic cell death associated with the disease. Here, we demonstrate the ability of gamma-glutamylcysteine ethyl ester, a lipid permeable derivative of the major substrate for scavenger glutathione synthesis, to counteract glutathione loss and neurodegeneration associated with in vitro and in vivo administration of MPTP or its derivatives. This data suggests that prevention of glutathione depletion is a likely therapeutic target for the disease.     

RETRACTED: Resibufogenin inhibited colorectal cancer cell growth and tumorigenesis through triggering ferroptosis and ROS production mediated by GPX4 inactivation

Resibufogenin (RB) has been used for cancer treatment, but the underlying mechanisms are still unclear. This study aimed to investigate the effects of RB treatment on colorectal cancer (CRC) cells, and to determine the underlying mechanisms. The cell counting kit-8 assay was used to determine cell viability. Cell morphology was observed under light microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was employed to detect cell apoptosis. Intracellular ferrous iron (Fe²⁺), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species levels were detected by using commercial iron assay kit, MDA assay kit, GSH assay kit, and 2,7-dichlorodihydrofluorescein diacetate probes, respectively. The protein expressions were determined by Western blot and immunohistochemistry. RB inhibited cell viability in the CRC cell lines (HT29 and SW480) in a dose- and time-dependent manner, and caused cytotoxicity to the normal colonic epithelial cell line (NCM460) at high dose. Similarly, RB induced morphological changes in CRC cells from normal to round shape, and promoted cell death. Of note, RB triggered oxidative stress and ferroptotic cell death in CRC cells, and only ferroptosis inhibitors (deferoxamine and ferrostatin-1), instead of inhibitors for other types of cell death (apoptosis, autophagy, and necroptosis), reversed the inhibitory effects of RB on CRC cell proliferation. Furthermore, glutathione peroxidase 4 (GPX4) was inactivated by RB treatment, and overexpression of GPX4 alleviated RB-induced oxidative cell death in CRC cells. Consistently, the in vivo experiments validated that RB also triggered oxidative stress, and inhibited CRC cells growth and tumorigenicity in mice models. RB can inhibit CRC cells growth and tumorigenesis by triggering ferroptotic cell death in a GPX4 inactivation-dependent manner.     

Ceramides modulate programmed cell death in plants

The balance between the bioactive sphingolipid ceramide and its phosphorylated derivative has been proposed to modulate the amount of programmed cell death (PCD) in eukaryotes. We characterized the first ceramide kinase (CERK) mutant in any organism. The Arabidopsis CERK mutant, called accelerated cell death 5, accumulates CERK substrates and shows enhanced disease symptoms during pathogen attack and apoptotic-like cell death dependent on defense signaling late in development. ACD5 protein shows high specificity for ceramides in vitro. Strikingly, C2 ceramide induces, whereas its phosphorylated derivative partially blocks, plant PCD, supporting a role for ceramide phosphorylation in modulating cell death in plants.     

A mechanism for glutamate toxicity in the C6 glioma cells involving inhibition of cystine uptake leading to glutathione depletion.

We have demonstrated that addition of L-glutamate in millimolar amounts to a culture of C6 glioma cells induced cell death within 24 h. The glutamate-induced toxicity in the C6 glioma cells was completely suppressed by adding L-cystine (0.4-1.0 mM), while the C6 cells degenerated in L-cystine-deprived culture medium. Kinetic studies of [35S]cystine and [3H]glutamate uptake showed that cystine competitively inhibited glutamate uptake, and conversely glutamate inhibited cystine uptake competitively, suggesting that C6 cells have a cystine/glutamate antiporter (system CG or Xc) similar to that already described in the periphery. Exogenous cystine (1 mM) stimulated a release of endogenous glutamate from C6 cells in a Na(+)-independent Cl(-)-dependent fashion. Thus, the antiporter normally transports glutamate out of and cystine into the cells. With the glutamate analogues tested, there was a good correlation between cytotoxicity and inhibition of cystine uptake. The de novo synthesis of glutathione was largely dependent upon the uptake of extracellular cystine. Intracellular levels of glutathione were dramatically decreased within 8-10 h by culture in glutamate-added or cystine-free medium. Vitamin E (100 microM), an antioxidant, rescued the death of C6 cells induced by glutamate exposure or by culture in cystine-deprived medium, but did not restore the apparent decrease of intracellular glutathione. Taken together, the present data strongly indicate that glutamate-induced cell death is initially due to inhibition of cystine uptake through the antiporter Xc system; such inhibition leads to glutathione depletion exposing the cells to oxidative stress. Excess of extracellular glutamate introduced from endogenous or exogenous roots might disorder this mechanism, resulting in cell death.     

Techniques used to define human MHC antigens: serology

The classical, routine test employed for definition of HLA antigens expressed in humans (tissue typing) is the complement-mediated cytotoxicity assay developed by Terasaki and McClelland in the early 1960s. In both healthy persons and patients, the assay target cells are usually lymphocytes obtained from peripheral blood, but when typing cadaver donors, splenic or lymph node lymphocytes can be used. HLA-A, B, Cw (class I) antigens are expressed on all nucleated cells while HLA-DR, DQ (class II) are restricted to B lymphocytes and immune activated cells. Tissue typing has been achieved using culture cells from amniocentesis and typing of cell lines is possible with small modifications to the standardised cytotoxicity assay. Usually, target cells are incubated under oil with typing antisera at 22 degrees C in a 60- or 72-well Terasaki tray. After 30 min rabbit serum is added as a source of complement. After a further 60 min incubation the test is stained. A positive reaction results in target cell death. There are local variations to this test. Automation of the assay is now commonplace, from reagent dispensing to automated reading of finished assay. The use of antibody-coated magnetisable microspheres has enabled separation of pure B lymphocyte samples for class II typing and has reduced incubation times through antigen modulation. It is possible to define antibodies to HLA antigens in the same assay using target cells with known HLA phenotypes.     

The use of nitroxide radical-containing nanoparticles coupled with piperine to protect neuroblastoma SH-SY5Y cells from Aβ-induced oxidative stress

The antioxidant effect and potential mechanism of nitroxide radical-containing nanoparticles (RNPs) coupled with piperine (PI) were investigated in human neuroblastoma SH-SY5Y cells. The effects of RNP/PI on SH-SY5Y cell lines was determined by WST assay for cell viability, nitroblue tetrazolium and deoxyribose assay for reactive oxygen species generation, ELISA assay for reactive oxygen species products and apoptotic cell death, and biochemical techniques for catalase and glutathione peroxidase activity. The RNP/PI significantly reduced the reactive oxygen species level and reactive oxygen species products compared with those of cells treated with RNPs alone. The RNP/PI treatment enhanced catalase and glutathione peroxidase activity. The combination of RNP/PI has been found to have an augmented antioxidant effect on an Alzheimer's model in vitro. The mechanism of the protective effect of this combination therapy was correlated in this study with its ability to reduce the generation of reactive oxygen species and prevent apoptosis via scavenging enzyme action pathways.     

Quantitation of reduced and total glutathione at the femtomole level by high-performance liquid chromatography with fluorescence detection: application to red blood cells and cultured fibroblasts

A new rapid and highly sensitive HPLC method with ortho-phthalaldehyde (OPA) pre-column derivatization has been developed for determination of reduced glutathione (GSH) and total glutathione (GSHt) in human red blood cells and cultured fibroblasts. OPA derivatives are separated on a reversed-phase HPLC column with an acetonitrile-sodium acetate gradient system and detected fluorimetrically. An internal standard (glutathione ethyl ester) is added to facilitate quantitation. Total glutathione is determined after reduction of disulfide groups with dithiothreitol; the oxidized glutathione (GSSG) concentration is calculated by subtraction of the GSH level from the GSHt level. The assay shows high sensitivity (50 fmol per injection, the lowest reported), good precision (C.V. <5.0%), an analytical recovery of GSH and GSSG close to 100%, and linearity (r > 0.999). This HPLC technique is very simple and rapid. Its wide applicability and high sensitivity make it a convenient and reliable method for glutathione determination in various biological samples.     

Bioanalysis of m-iodobenzylguanidine in plasma by high-performance liquid chromatography after derivatization with benzoin.

A sensitive chromatographic assay has been developed for m-iodobenzylguanidine (MIBG) in human plasma based on the derivatization with benzoin. MIBG is first isolated from plasma using solid-phase extraction on a cyanopropyl-modified silica phase. After evaporation of the eluate, a fluorescent derivative is formed using benzoin. The derivative is analysed by reversed-phase liquid chromatography using a mixture 60% (v/v) acetonitrile, 30% (v/v) water and 10% (v/v) of the 0.5 M Tris buffer (pH 8.0) as the eluent and fluorescence detection at 320 nm for excitation and 435 nm for emission, respectively. In the evaluated concentration range (2-200 ng/ml) precisions < or = 10% and accuracies in between 90 and 100% have been found, with 2 ng/ml being the lower limit of quantification using a 0.5-ml plasma sample volume. The assay can also be used without the internal standard benzylguanidine. The assay was successfully used to obtain a pharmacokinetic curve of MIBG.     

The inhibition of death receptor mediated apoptosis through lysosome stabilization following internalization of carboxyfullerene nanoparticles

Cells undergo apoptosis through two major pathways, the extrinsic pathway (death receptor pathway) and the intrinsic pathway (the mitochondrial pathway). It is well known that nanomaterials of water- soluble fullerene derivatives are potent antioxidants and help to prevent the overproduction of mitochondrial reactive oxygen species (ROS). However, whether their interaction with cells via the death receptor pathway is direct or indirect remains poorly understood. Here, we show that a bis-adduct malonic acid derivative of fullerene, C??(C(COOH)?)?, inhibits tumor necrosis factor alpha-initiated cellular apoptosis via stabilizing lysosomes. Data presented here demonstrate that nano-sized aggregates of this water-soluble fullerene derivative are endocytosed into cells and enriched in the lysosomes. During the internalization of C??(C(COOH)?)?, the expression of Hsp 70 is significantly upregulated, promoting cell survival by inhibiting the permeabilization of lysosomal membranes. In addition, the acidic environment inside lysosomes has a marked but temporary effect on the size distribution of fullerenic nanoparticles, and may disperse the aggregated C??(C(COOH)?)? nanoparticles into single molecules or smaller aggregates. These single molecules or smaller aggregates may insert into the lysosomal membranes, further stabilizing them and decreasing the release of cathepsins from lysosomes, leading to the inhibition of tumor necrosis factor-induced apoptosis. C??(C(COOH)?)? nanoparticles can thus protect cells by stabilizing lysosomal membranes via both upregulated expression of Hsp 70 and by their interactions with lysosomal membranes.     

Genotoxicity, cytotoxicity, and reactive oxygen species induced by single-walled carbon nanotubes and C(60) fullerenes in the FE1-Mutatrade markMouse lung epithelial cells

Viability, cell cycle effects, genotoxicity, reactive oxygen species production, and mutagenicity of C(60) fullerenes (C(60)) and single-walled carbon nanotubes (SWCNT) were assessed in the FE1-Mutatrade markMouse lung epithelial cell line. None of these particles induced cell death within 24 hr at doses between 0 and 200 microg/ml or during long-term subculture exposure (576 hr) at 100 microg/ml, as determined by two different assays. However, cell proliferation was slower with SWCNT exposure and a larger fraction of the cells were in the G1 phase. Exposure to carbon black resulted in the greatest reactive oxygen species generation followed by SWCNT and C(60) in both cellular and cell-free particle suspensions. C(60) and SWCNT did not increase the level of strand breaks, but significantly increased the level of FPG sensitive sites/oxidized purines (22 and 56%, respectively) determined by the comet assay. The mutant frequency in the cII gene was unaffected by 576 hr of exposure to either 100 microg/ml C(60) or SWCNT when compared with control incubations, whereas we have previously reported that carbon black and diesel exhaust particles induce mutations using an identical exposure scenario. These results indicate that SWCNT and C(60) are less genotoxic in vitro than carbon black and diesel exhaust particles.     

A sensitive and specific assay for glutathione with potential application to glutathione disulphide, using high-performance liquid chromatography-tandem mass spectrometry

We have utilised the combination of sensitivity and specificity afforded by coupling high-performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS-MS) to produce an assay which is suitable for assaying glutathione (GSH) concentrations in liver tissue. The sensitivity suggests it may also be suitable for extrahepatic tissues. The method has been validated for GSH using mouse liver samples and also allows the assay of GSSG. The stability of GSH under conditions relevant to the assay has been determined. A 20-microl amount of a diluted methanol extract of tissue is injected with detection limits of 0.2 pmol for GSH and 2 pmol for GSSG. The HPLC uses an Altima C18 (150 x 4.6 mm, 5 microm) column at 35 degrees C. Chromatography utilises a linear gradient from 0 to 10% methanol in 0.1% formic acid over 5 min, with a final isocratic stage holding at 10% methanol for 5 min. Total flow rate is 0.8 ml/min. The transition from the M+H ion (308.1 m/z for GSH, and 613.3 m/z for GSSG) to the 162.0 m/z (GSH) and 355.3 m/z (GSSG) fragments are monitored.     

Olfactory epithelium as a novel toxic target following an intravenous administration of vincristine to mice

To delineate morphological characteristics of olfactory lesions induced by vincristine (VCR), a vinca alkaloid derivative with antitumor activity, male BALB/c mice were given a single intravenous injection of 1.95 mg/kg, an estimated 10% lethal dose (designated as day 1). The animals were serially sacrificed on days 2, 3, 5, 10, 15 and 60, and the nasal mucosa was examined histopathologically. Cell death was noted in the olfactory epithelia adjacent to the respiratory epithelia from days 2 to 5. Inflammatory responses were not detected throughout the observation periods. Cell death was identified as apoptotic by the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay and electron microscopy. Mitotic figures and proliferating cell nuclear antigen (PCNA)-positive reactions were diffusely scattered in both the basal and sensory cells. On days 10 or after, no prominent histological abnormalities were noted in the olfactory epithelia, which suggests the aforementioned lesions were completely recovered. These results demonstrate that it is essential to perform histopathological evaluation of the nasal mucosa during an early preclinical stage for novel antitumor drugs, since olfactory lesions due to the certain compounds like VCR may not be detected by any other procedure.     

Heat shock treatment protects human HL-60 cells from apoptosis induced by lectin II isolated from Korean mistletoe, Visum album var. Coloratum

Mistletoe lectin II (ML II) isolated from Korean mistletoe (Viscum album var. Coloratum), an effective therapeutic agent for cancers, is known to induce cell death via apoptosis. In the present study, we found the protective effect of heat shock treatment of human leukemia HL-60 cells against ML II-induced apoptosis. Exposure of HL-60 cells to ML II for 4 h resulted in apoptosis of the cells, which was evaluated by examining “DNA ladder” formation and DNA fragmentation assay. The DNA fragmentation was significantly reduced in the cells subjected to heat shock treatment by incubation at 42 °C for 1 h and subsequently allowed to recover for 2-16 h at 37 °C., prior to exposure to ML II. HL-60 cells transfected with heat shock protein (hsp) 70 gene exhibited resistance to ML II-induced apoptosis very similar to that seen when untransfected cells were heat-shocked. These results indicate that ML II-induced apoptosis in HL-60 cells is inhibited by heat shock treatment, at least in part, via a hsp 70-mediated mechanism.     

Neuroprotective and antiinflammatory properties of a novel demethylated curcuminoid

A demethylated derivative of curcumin (DC; 67.8% bisdemethylcurcumin, 20.7% demethylmonodemethoxycurcumin, 5.86% bisdemethoxycurcumin, 2.58% demethylcurcumin) was prepared by using a 95% extract of curcumin (C(95); 72.2% curcumin, 18.8% monodemethoxycurcumin, 4.5% bisdemethoxycurcumin). DC increased glutathione and reduced reactive oxygen species (ROS) in HT4 neuronal cells. In a model of glutamate-induced death of HT4, DC was more effective than C(95) in neuroprotection. The protective effects of DC were retained even when DC was withdrawn from culture media after pretreatment. DC treatment, unlike an equal dose of C(95), completely spared glutamate-induced loss of cellular GSH. Both DC and C(95) prevented glutamate-induced elevation of cellular ROS but failed to attenuate glutamate-induced elevation of intracellular calcium. In human microvascular endothelial cells (HMECs) challenged with TNF-alpha, GeneChip analysis revealed that only a subcluster of 23 TNF-alpha-inducible genes were uniquely sensitive to C(95). In sharp contrast, 1,065 TNF-alpha-inducible genes were sensitive to DC but not to C(95), suggesting that DC was more effective in antagonizing the effects of TNF-alpha on HMECs. Functional analysis identified that the genes uniquely sensitive to DC belonged in four functional categories: cytokine-receptor interaction, focal adhesion, cell adhesion, and apoptosis. Real-time PCR as well as ELISA studies demonstrated that TNF-alpha-inducible CXCL10 and CXCL11 expression was sensitive to DC but not to C(95). Flow-cytometry studies recognized ICAM-1 and VCAM-1 as TNF-alpha-inducible adhesion molecules that were uniquely sensitive to DC. Taken together, DC exhibited promising neuroprotective and antiinflammatory properties that must be characterized in vivo.     

Gas chromatographic-mass spectrometric analysis of stable isotopes of cysteine and glutathione in biological samples.

A gas chromatographic-mass spectrometric (GC-MS) procedure for the determination of stable isotope labelled glutathione has been applied to animal and human samples. The method, based on preparation of the N,S-ethoxycarbonyl methyl ester derivative of the intact peptide, is rapid and requires little or minor tissue treatment. The same method was applied to cysteine. The method was found to be reliable in terms of within-day and between-day precision, accuracy and linearity. The procedure was applied in humans and animals to determine in vivo the glutathione fractional synthesis rate using labelled cysteine infusion. The glutathione fractional synthesis rate was found to be 22.5%/day in blood from a healthy volunteer and 337+/-29%/day in rat liver.     

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

Background  

Cellular 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.     

Immunophilin ligands prevent H2O2-induced apoptotic cell death by increasing glutathione levels in neuro 2A neuroblastoma cells

We examined the effects of FK506 and its non-immunosuppressive derivative, GPI1046, on H2O2-induced reduction of cell viability and apoptotic cell death in Neuro 2A cells. Our results suggest that the protective properties of GPI1046 against H2O2-induced reduction of cell viability are equipotent with those of FK506 and may be mediated by increased intracellular concentrations of glutathione (GSH). In addition, both FK506 and GPI1046 prevented apoptotic cell death in Neuro 2A cells, although the antiapoptotic effect of FK506 was somewhat stronger than that of GPI1046. These findings suggest that non-immunosuppressive immunophilin ligands such as GPI1046 might be potentially useful in treatment of neurodegenerative diseases without serious side effects such as immune deficiency.     

Immobilization of Candida rugosa lipase on magnetized Dacron: kinetic study

Candida rugosa lipase has been covalently immobilized on ferromagnetic azide polyethyleneterepthalate (Dacron) with specific activity retention of 16% for 4-nitrophenyl palmitate and 24% for hydrolysis of triolein in hexane. The immobilized enzyme was more thermal stable than the soluble one, retaining 78.8% of the activity after 1 h at 60 degrees C. Also, this immobilized derivative was stable at the storage at 4 degrees C. It has been used 5 cycles for pNPP hydrolysis without loss of activity. Soluble and immobilized Candida rugosa lipase showed a Michaelian behavior for fatty acid 4-nitrophenyl esters and different apparent K(M) values: 0.110 mM and 0.124 mM (4-nitrophenyl palmitate - C16); 0.193 mM and 0.235 mM (4-nitrophenyl laurate - C12) and 0.206 mM and 0.119 mM (4-nitrophenyl butyrate - C4), respectively. The immobilized lipase was more efficient for catalyzing the hydrolysis of 4-nitrophenyl esters with short chain length fatty acid (4-NPB - C4) than soluble enzyme. The ferromagnetic Dacron-lipase derivative was able to catalyze the synthesis of triolein from glycerol and oleic acid with 50% of conversion after 72 h at 40 degrees C.