Presence Of Buthionine Sulfoximine Research Articles

SOD mimetic cerium oxide nanorods protect human hepatocytes from oxidative stress

Recently, cerium oxide nanoparticles (CeNPs) have emerged as potential materials for several biomedical applications due to their excellent biocompatibility and biological enzyme-like activities. The mechanism of observed antioxidant (superoxide dismutase and catalase) enzyme-like activities of spherical-shaped CeNPs is suggested due to the switching between its two oxidation states (3+ and 4+). The shape of nanoparticles plays a significant role in controlling the catalytic activity; therefore, we have studied the antioxidant enzyme-like activities of cerium oxide nanorods. Our results suggest that CeNPs (rods) display excellent SOD-like activity as well as auto regeneration ability of its surface “Ce” atoms, comparable to CeNPs (spheres). The cytotoxicity and genotoxicity studies of CeNPs (rods) showed non-toxicity to WRL-68 (human hepatocyte) cells and impart protection from the harmful effects of reactive oxygen species generated in the presence of Buthionine sulfoximine (BSO). BSO exposure induces inhibition of glutathione (GSH) synthesis that regulates the redox status of the cytosol. The cellular internalization of CeNPs (rods) in WRL-68 cells revealed quick uptake by the cells in a time and concentration-dependent manner. The surface regeneration property of CeNPs (rods) was analysed in the presence of hydrogen peroxide (H2O2). Results suggested that the atoms present on the surface of CeNPs (rods) were oxidised into “4+” states after the addition of H2O2. After incubation of 15 days, they regained their original “3+” oxidation state by regenerating their surface, similar to spherical nanoparticles.

Abstract 5485: The combined effects of polyamine and GSH inhibition on the viability of human cancer cell lines

Abstract Polyamines play essential roles in DNA stability and cell proliferation, making polyamine metabolism a potential target for cancer therapeutics. The spermine analog N1,N11- Diethylnorspermine (DenSpm) reduces levels of ODC, the enzyme responsible for polyamine biosynthesis, and also induces the enzyme SSAT, responsible for the catabolism of polyamines. Polyamines have been shown to inactivate reactive oxygen species (ROS), including peroxides, protecting mammalian cells from oxidative damage to nucleic acids. The catabolism of polyamines by SSAT, in contrast, leads to the production of ROS, which results in cellular death and limits the progression of cancer. Buthionine sulfoximine (BSO) selectively depletes stores of intracellular glutathione GSH, an antioxidant that neutralizes ROS. We hypothesized that treating cancer cell lines with both DenSpm and BSO will have a synergistic cytotoxic effect on the cells, due to the depletion of ROS-scavenging polyamines, the generation of ROS caused by DenSpm and the absence of GSH. Treatment of the four cell lines, MCF-7, MDA-MD-231, Caco-2 and PANC-1, with DenSpm resulted in moderate reductions in cell viability as measured using the Cell Titer Blue assay (Promega, Inc.). As has been widely reported in other mammalian systems, DenSpm (10 µM) reduced intracellular polyamine concentrations (HPLC of benzoylated polyamine derivatives) and induced SSAT (Western Blots). Treatment of cells with BSO (500 µM) caused a significant decrease in intracellular GSH levels. Similar findings have been reported in previous studies with human neuroblastoma lines (Anderson, et al. 1999). The combination of BSO and DenSpm had effects markedly greater than either compound alone. Pretreatment of cells with BSO (500 µM) for 24 hours with subsequent removal of BSO before DenSpm (10, 100, 1000 µM) addition resulted in significantly reduced viability in all cell lines. DenSpm was found to be most effective in the continued presence of BSO as compared to a 24-hour BSO pretreatment. The compounds used in combination also enhanced ROS production, further demonstrating their synergistic effects. Citation Format: John Grady, Davey Holzer, Kaci Keleher, Emma Meetz, Russ Feirer. The combined effects of polyamine and GSH inhibition on the viability of human cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5485. doi:10.1158/1538-7445.AM2017-5485

The diverse roles of glutathione-associated cell resistance against hypericin photodynamic therapy

The diverse responses of different cancers to treatments such as photodynamic therapy of cancer (PDT) have fueled a growing need for reliable predictive markers for treatment outcome. In the present work we have studied the differential response of two phenotypically and genotypically different breast adenocarcinoma cell lines, MCF7 and MDA-MB-231, to hypericin PDT (HYP-PDT). MDA-MB-231 cells were 70% more sensitive to HYP PDT than MCF7 cells at LD50. MCF7 were found to express a substantially higher level of glutathione peroxidase (GPX4) than MDA-MB-231, while MDA-MB-231 differentially expressed glutathione-S-transferase (GSTP1), mainly used for xenobiotic detoxification. Eighty % reduction of intracellular glutathione (GSH) by buthionine sulfoximine (BSO), largely enhanced the sensitivity of the GSTP1 expressing MDA-MB-231 cells to HYP-PDT, but not in MCF7 cells. Further inhibition of the GSH reduction however by carmustine (BCNU) resulted in an enhanced sensitivity of MCF7 to HYP-PDT. HYP loading studies suggested that HYP can be a substrate of GSTP for GSH conjugation as BSO enhanced the cellular HYP accumulation by 20% in MDA-MB-231 cells, but not in MCF7 cells. Studies in solutions showed that L-cysteine can bind the GSTP substrate CDNB in the absence of GSTP. This means that the GSTP-lacking MCF7 may use L-cysteine for xenobiotic detoxification, especially during GSH synthesis inhibition, which leads to L-cysteine build-up. This was confirmed by the lowered accumulation of HYP in both cell lines in the presence of BSO and the L-cysteine source NAC. NAC reduced the sensitivity of MCF7, but not MDA-MB-231, cells to HYP PDT which is in accordance with the antioxidant effects of L-cysteine and its potential as a GSTP substrate. As a conclusion we have herein shown that the different GSH based cell defense mechanisms can be utilized as predictive markers for the outcome of PDT and as a guide for selecting optimal combination strategies.

Characterization of cadmium chloride-induced BiP accumulation in Xenopus laevis A6 kidney epithelial cells

Endoplasmic reticulum (ER) stress can result in the accumulation of unfolded/misfolded protein in the ER lumen, which can trigger the unfolded protein response (UPR) resulting in the activation of various genes including immunoglobulin-binding protein (BiP; also known as glucose-regulated protein 78 or HSPA5). BiP, an ER heat shock protein 70 (HSP70) family member, binds to unfolded protein, inhibits their aggregation and re-folds them in an ATP-dependent manner. While cadmium, an environmental contaminant, was shown to induce the accumulation of HSP70 in vertebrate cells, less information is available regarding the effect of this metal on BiP accumulation or function. In this study, cadmium chloride treatment of Xenopus laevis A6 kidney epithelial cells induced a dose- and time-dependent increase in BiP, HSP70 and heme oxygenase-1 (HO-1) accumulation. Exposure of cells to a relatively low cadmium concentration at a mild heat shock temperature of 30°C greatly enhanced BiP and HSP70 accumulation compared to cadmium at 22°C. Treatment of cells with the glutathione synthesis inhibitor, buthionine sulfoximine, enhanced cadmium-induced BiP and HSP70 accumulation. Immunocytochemistry revealed that cadmium-induced BiP accumulation occurred in a punctate pattern in the perinuclear region. In some cells treated with cadmium chloride or the proteasomal inhibitor, MG132, large BiP complexes were observed that co-localized with aggregated protein or aggresome-like structures. These BiP/aggresome-like structures were also observed in cells treated simultaneously with cadmium at 30°C or in the presence of buthionine sulfoximine. In amphibians, the association of BiP with unfolded protein and its possible role in aggresome function may be vital in the maintenance of cellular proteostasis.

Abstract 4792: The effects of KPT-330, a selective inhibitor of nuclear export, on nuclear topoisomerase II alpha levels and etoposide activity in human myeloid leukemia HL-60 cells

Abstract DNA topoisomerase IIá (topo II) is a known cargo protein for exportin 1 (XPO1, CRM1) (Seminars Cancer Biol. 27: 62-73, 2014) for nuclear export. Here we utilized KPT-330, a selective inhibitor of nuclear export, to evaluate its cytotoxicity, its effects on the nuclear content of topo II in human myeloid leukemia HL60 cells and on the subsequent activity of the topo II targeting agent etoposide. Since KPT-330 is a potential michael acceptor and is known to interact with the CRM1 active site Cys528 to inhibit its activity, we first analyzed interaction of KPT-330 with glutathione (GSH)-containing cysteine. In an in vitro binding study KPT-330 was weakly bound to GSH with a Kd = 130+/-24 μM. In contrast, the natural product compound parthenolide, also a michael acceptor, bound to GSH with 30-fold greater affinity. Depletion of cellular GSH with buthionine sulfoximine (BSO) did not significantly alter the 72 hr growth inhibitory effects of KPT-330 yielding I50-values of 109 +/- 19 and 80 +/- 44 nM (p = 0.133) in the absence and presence of BSO. In contrast, GSH depletion significantly enhanced the activity of parthenolide yielding I50-values of 2.59 +/- 0.16 and 1.35 +/- 0.15 μM (p<0.001) in the absence or presence of BSO. KPT-330 (50 μM) did not alter cellular GSH levels in HL60 cells. Using 3’-(p-hydroxyphenyl) fluorescein, KPT-330 was antioxidant in GSH replete and depleted HL60 cells. Together results indicate that conjugation with GSH does not play a significant role in KPT-330 activity. Less than additive cytotoxicity (trypan blue) and apoptosis (Hoescht) were observed using a single fixed ratio of KPT-330 and etoposide incubated either simultaneously or after overnight pre-incubation with KPT-330. Similarly, using the Chou and Talalay technique, KPT-330/etoposide combinations were less than additive in exponentially growing HL60 cells. The mechanism(s) for this apparent antagonism using this combination are under investigation. Using fluorescently labeled topo II antibody, nuclear content of exponentially growing HL60 cells was 85.9 +/- 2.3% of total in the absence of KPT-330 and 86.5+/- 1.8% after an 16 hr incubation with 100 nM KPT-330. In plateau phase cells there was a statistically significant decrease in nuclear topoisomerase IIá to 74.1+/-1.8% compared to exponentially growing cells (p<0.05). In these plateau phase cells, a similar 16 hr incubation with 100 nM KPT-330 resulted in a significant increase in nuclear topoisomerase IIá to 82.6+/-2.5% of total compared to controls (p<0.05). Results are in accord with the known CRM1-mediated shuttling of topo II from the nucleus only in cells approaching or in plateau phase (Exp. Cell Res. 313: 627-637, 2007). Studies are underway to evaluate etoposide-mediated DNA damage and topo II-covalent complexes in HL60 cells in various growth phases after KPT-330 treatment as a correlate with nuclear topo II residency. Citation Format: Erica Godley, Ragu Kanagasabai, Soumendrakrishna Karmahapatra, Jack C. Yalowich. The effects of KPT-330, a selective inhibitor of nuclear export, on nuclear topoisomerase II alpha levels and etoposide activity in human myeloid leukemia HL-60 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4792.

Resveratrol attenuates monocyte-to-macrophage differentiation and associated inflammation via modulation of intracellular GSH homeostasis: Relevance in atherosclerosis

Monocyte-to-macrophage differentiation promotes an inflammatory environment within the arterial vessel wall that causes a mal-adaptive immune response, which contributes to the progression of atheromatous plaque formation. In the current study, we show that resveratrol, a well-known antioxidant, dose-dependently attenuated phorbol myristate acetate (PMA)-induced monocyte-to-macrophage differentiation, as measured by cell adhesion, increase in cell size, and scavenger receptor expression in THP-1 monocytes. Also, resveratrol significantly inhibited PMA-induced pro-inflammatory cytokine/chemokine and matrix metalloprotease (MMP-9) production. This inhibitory effect of resveratrol on monocyte differentiation results from its ability to restore intracellular glutathione (GSH) status, as resveratrol in the presence of buthionine sulfoximine (BSO) failed to affect monocyte differentiation. Furthermore, PMA-induced monocyte differentiation and inflammation was greatly inhibited when cells were co-treated with N-Acetyl-l-cysteine (NAC), a GSH precursor, while the presence of BSO aggravated these processes. These results also show that resveratrol mediated up-regulation of GSH is due to AMP-activated protein kinase (AMPK)-α activation, as compound C (AMPK inhibitor) treatment drastically depleted intracellular GSH and exacerbated PMA-induced monocyte differentiation and pro-inflammatory cytokine production. More importantly, chronic administration of resveratrol efficiently prevented monocyte infiltration and markedly diminished angiotensin (Ang)-II-induced atheromatous plaque formation in apolipoprotein-E knockout (ApoE−/−) mice. We conclude that, intracellular GSH status plays a critical role in regulating monocyte-to-macrophage differentiation and inflammation and resveratrol, by restoring GSH levels, inhibits these processes. Taken together, these results suggest that resveratrol can attenuate atherosclerosis, at least, in part, by inhibiting monocyte differentiation and pro-inflammatory cytokines production.

Elevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver.

Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule.

CYP2E1 induction leads to oxidative stress and cytotoxicity in glutathione-depleted cerebellar granule neurons

Increasing evidence suggests that brain cytochrome P450 (CYP) can contribute to the in situ metabolism of xenobiotics. In the liver, some xenobiotics can be metabolized by CYPs into more reactive products that can damage hepatocytes and induce cell death. In addition, normal CYP activity may produce reactive oxygen species (ROS) that contribute to cell damage through oxidative mechanisms. CYP2E1 is a CYP isoform that can generate ROS leading to cytotoxicity in multiple tissue types. The aim of this study was to determine whether CYP2E1 induction may lead to significant brain cell impairment. Immunological analysis revealed that exposure of primary cerebellar granule neuronal cultures to the CYP inducer isoniazid, increased CYP2E1 expression. In the presence of buthionine sulfoximine, an agent that reduces glutathione levels, isoniazid treatment also resulted in reactive oxygen species (ROS) production, DNA oxidation and cell death. These effects were attenuated by simultaneous exposure to diallyl sulfide, a CYP2E1 inhibitor, or to a mimetic of superoxide dismutase/catalase, (Euka). These results suggest that in cases of reduced antioxidant levels, the induction of brain CYP2E1 could represent a risk of in situ neuronal damage.

BIMELis a key effector molecule in oxidative stress-mediated apoptosis in acute myeloid leukemia cells when combined with arsenic trioxide and buthionine sulfoximine

sBackgroundArsenic trioxide (ATO) is reported to be an effective therapeutic agent in acute promyelocytic leukemia (APL) through inducing apoptotic cell death. Buthionine sulfoximine (BSO), an oxidative stress pathway modulator, is suggested as a potential combination therapy for ATO-insensitive leukemia. However, the precise mechanism of BSO-mediated augmentation of ATO-induced apoptosis is not fully understood. In this study we compared the difference in cell death of HL60 leukemia cells treated with ATO/BSO and ATO alone, and investigated the detailed molecular mechanism of BSO-mediated augmentation of ATO-induced cell death.MethodsHL60 APL cells were used for the study. The activation and expression of a series of signal molecules were analyzed with immunoprecipitation and immunoblotting. Apoptotic cell death was detected with caspases and poly (ADP-ribose) polymerase activation. Generation of intracellular reactive oxygen species (ROS) was determined using a redox-sensitive dye. Mitochondrial outer membrane permeabilization was observed with a confocal microscopy using NIR dye and cytochrome c release was determined with immunoblotting. Small interfering (si) RNA was used for inhibition of gene expression.ResultsHL60 cells became more susceptible to ATO in the presence of BSO. ATO/BSO-induced mitochondrial injury was accompanied by reduced mitochondrial outer membrane permeabilization, cytochrome c release and caspase activation. ATO/BSO-induced mitochondrial injury was inhibited by antioxidants. Addition of BSO induced phosphorylation of the pro-apoptotic BCL2 protein, BIMEL, and anti-apoptotic BCL2 protein, MCL1, in treated cells. Phosphorylated BIMEL was dissociated from MCL1 and interacted with BAX, followed by conformational change of BAX. Furthermore, the knockdown of BIMEL with small interfering RNA inhibited the augmentation of ATO-induced apoptosis by BSO.ConclusionsThe enhancing effect of BSO on ATO-induced cell death was characterized at the molecular level for clinical use. Addition of BSO induced mitochondrial injury-mediated apoptosis via the phosphorylation of BIMEL and MCL1, resulting in their dissociation and increased the interaction between BIMEL and BAX.

The influence of glutathione on redox regulation by antioxidant proteins and apoptosis in macrophages exposed to 2-hydroxyethyl methacrylate (HEMA)

Resin monomers like 2-hydroxyethyl methacrylate (HEMA) disturb cell functions including responses of the innate immune system, mineralization and differentiation, or induce cell death via apoptosis. These phenomena are associated with oxidative stress and a reduction in the concentration of the antioxidant glutathione (GSH), resulting in imbalanced redox homeostasis. Thus far, the precise mechanism of how resin monomers interfere with cellular redox regulation is unknown. The present study provides insight into the induction of apoptosis and the differential expression of antioxidant enzymes depending on the availability of GSH. Buthionine sulfoximine (BSO) was used to inhibit GSH synthesis, while 2-oxothiazolidine-4-carboxylate (OTC), and N-acetylcysteine (NAC) as prodrugs supported GSH synthesis in RAW264.7 mouse macrophages exposed to HEMA (0–8 mm) for 24 h. The level of GSH was significantly decreased after cells were preincubated with BSO, and the formation of reactive oxygen species (ROS) increased in cultures subsequently exposed to HEMA. Apoptosis was drastically increased by BSO in HEMA-exposed cell cultures as well, but OTC and NAC retracted HEMA-induced cell death. These results show that dental monomer-induced apoptosis is causally related to the availability of GSH. The hydrogen peroxide decomposing enzymes glutathione peroxidase (GPx1/2) and catalase were differentially regulated in HEMA-exposed cultures. Expression of GPx1/2 was inhibited by HEMA and further reduced in the presence of BSO. SOD1 (superoxide dismutase) expression was inhibited in the presence of HEMA, and was decreased to an even greater extent by BSO, possibly due to H2O2-feedback inhibition. The expression of catalase was considerably up-regulated in HEMA-exposed cultures, implying that H2O2 is the type of ROS that is significantly increased in monomer-exposed cells. OTC and NAC counteracted the effect of HEMA on GPx1/2, SOD1, and catalase expression. HO-1 (heme oxygenase) expression was strongly enhanced by HEMA, suggesting the need for further antioxidants like bilirubin to support enzyme activities that directly regulate H2O2 equilibrium. Expression of the oxidoreductase thioredoxin (TRX1), the second major thiol-dependent antioxidant system in eukaryotic cells, was slightly reduced, while the oxygen-sensing protein HIF-1α was downregulated in HEMA-exposed cell cultures. These results indicate that cells and tissues actively respond to monomer-induced oxidative stress by the differential expression of enzymatic antioxidants.

The significance of glutathione for photoprotection at contrasting temperatures in the alpine plant species Soldanella alpina and Ranunculus glacialis

The significance of total glutathione content was investigated in two alpine plant species with highly differing antioxidative scavenging capacity. Leaves of Soldanella alpina and Ranunculus glacialis incubated for 48 h in the presence of buthionine-sulfoximine had 50% lower glutathione contents when compared with leaves incubated in water. The low leaf glutathione content was not compensated for by activation of other components involved in antioxidative protection or electron consumption. However, leaves with normal but not with low glutathione content increased their ascorbate content during high light (HL) treatment (S. alpina) or catalase activity at low temperature (LT) (R. glacialis), suggesting that the mere decline of the leaf glutathione content does not act as a signal to ameliorate antioxidative protection by alternative mechanisms. CO(2)-saturated oxygen evolution was not affected in glutathione-depleted leaves at various temperatures, except at 35°C, thereby increasing the high temperature (HT) sensitivity of both alpine species. Leaves with low and normal glutathione content were similarly resistant to photoinhibition and photodamage during HL treatment at ambient temperature in the presence and absence of paraquat or at LT. However, HL- and HT-induced photoinhibition increased in leaves with low compared to leaves with normal glutathione content, mainly because the recovery after heat inactivation was retarded in glutathione-depleted leaves. Differences in the response of photosystem II (PSII) activity and CO(2)-saturated photosynthesis suggest that PSII is not the primary target during HL inactivation at HT. The results are discussed with respect to the role of antioxidative protection as a safety valve for temperature extremes to which plants are not acclimated.

Promising effects of the 4HPR–BSO combination in neuroblastoma monolayers and spheroids

To enhance the efficacy of fenretinide (4HPR)-induced reactive oxygen species (ROS) in neuroblastoma, 4HPR was combined with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, in neuroblastoma cell lines and spheroids, the latter being a three-dimensional tumor model. 4HPR exposure (2.5–10 μM, 24 h) resulted in ROS induction (114–633%) and increased GSH levels (68–120%). A GSH depletion of 80% of basal levels was observed in the presence of BSO (25–100 μM, 24 h). The 4HPR–BSO combination resulted in slightly increased ROS levels (1.1- to 1.3-fold) accompanied by an increase in cytotoxicity (110–150%) compared to 4HPR treatment alone. A correlation was observed between the ROS-inducing capacity of each cell line and the increase in cytotoxicity induced by 4HPR–BSO compared to 4HPR. No significant correlation between baseline antioxidant levels and sensitivity to 4HPR or BSO was observed. In spheroids, 4HPR–BSO induced a strong synergistic growth retardation and induction of apoptosis. Our data show that BSO increased the cytotoxic effects of 4HPR in neuroblastoma monolayers and spheroids in ROS-producing cell lines. This indicates that the 4HPR–BSO combination might be a promising new strategy in the treatment of neuroblastoma.

Identification of a Pentatricopeptide Repeat Protein Implicated in Splicing of Intron 1 of Mitochondrial nad7 Transcripts

Splicing of plant organellar transcripts is facilitated by members of a large protein family, the pentatricopeptide repeat proteins. We have identified a pentatricopeptide repeat protein in a genetic screen for mutants resistant to inhibition of root growth by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis and consequently named BIR6 (BSO-insensitive roots 6). BIR6 is involved in splicing of intron 1 of the mitochondrial nad7 transcript. Loss-of-function mutations in BIR6 result in a strongly reduced accumulation of fully processed nad7 transcript. This affects assembly of Complex I and results in moderate growth retardation. In agreement with disruption of Complex I function, the genes encoding alternative NADH oxidizing enzymes are induced in the mutant, and the mutant plants are less sensitive to mannitol and salt stress. Mutation in the BIR6 gene allowed normal root growth in presence of BSO and strongly attenuated depletion of glutathione content at these conditions. The same phenotype was observed with other mutants affected in function of Complex I, thus reinforcing the importance of Complex I function for cellular redox homeostasis.

Developmental potential of bovine oocytes following IVM in the presence of glutathione ethyl ester

Glutathione (GSH) is synthesised during oocyte maturation and represents the oocyte's main non-enzymatic defence against oxidative stress. Inadequate defence against oxidative stress may be related to poor embryo quality and viability. In the present study, bovine oocytes were matured in vitro in the presence of GSH ethyl ester (GSH-OEt), a cell permeable GSH donor, and its effects on subsequent fertilisation and embryo development were assessed. GSH-OEt significantly increased the GSH content of IVM oocytes without affecting fertilisation or Day 3 cleavage rates. Maturation in the presence of GSH-OEt did not significantly increase the blastocyst rate compared with control oocytes. However, 5 mM GSH-OEt treatment resulted in significantly higher blastocyst total cell number. The GSH level of IVM oocytes was significantly decreased in the absence of cumulus cells and when cumulus-oocyte complexes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine oocytes and restored the rate of normal fertilisation, but not embryo development, to levels seen in control oocytes. Thus, GSH-OEt represents a novel approach for effective in vitro elevation of bovine oocyte GSH and improvement in blastocyst cell number.

The Diterpenoid Lactone Andrographolide (ANDRO) Stimulates Autophagy and Induces Reactive Oxygen Species (ROS)-Dependent Apoptosis in Lymphoma Cell Lines

ANDRO is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an important herbal medicine used in China. It has been reported to have anti-inflammatory, anti-hypertensive, anti-viral and immunostimulant properties. It has also been shown to inhibit cancer cell proliferation and induce apoptosis in HL-60 (leukemia), PC-3 (prostatic adenocarcinoma), MDA-MB-231 (breast cancer), HepG2 (liver cancer), HeLa (cervical cancer) and HCT116 (colorectal cancer) cell lines. The diterpenoids have been found to generate ROS and may increase apoptosis by altering the cellular redox state. We hypothesized that ANDRO would lead to cell death in lymphoma cell lines and that the effect may be related to altered cellular redox state. We studied the Burkitt p53 mutated Ramos cell line, the mantle cell lymphoma line Granta and L428, a resistant EBV-negative Hodgkin lymphoma cell line. We found that after incubation with increasing concentrations of ANDRO, there was dose and time-dependent cell death as measured by MTT. The IC50 (concentration that achieved 50% cell proliferation inhibition) at 48h was 20μM for Ramos, 40μM for Granta, and 50μM for L428. ROS was measured by oxidation of 2'7'dichlorofluorescein diacetate (DCFDA) to dichlorofluorescein (DCF) and analyzed by fluorescence-activated cell sorting (FACS) following incubation at 1hour (h), 2h, 3h, 5h, 38h, and 48h with ANDRO (20–80μM). ANDRO increased ROS production in all lymphoma cell lines, which was abrogated by the antioxidant N-acetyl-L-cysteine (NAC). Maximum ROS generation with ANDRO was seen at 48h for Ramos (1.7 fold), 5h for Granta (1.6 fold), and 38h for L428 (2.4 fold). To determine the mechanism of cell death, we measured apoptosis by Annexin-V/propidium iodide (PI), and detected by flow cytometry (FACS). Cells were treated with ANDRO in the presence or absence of the reduced glutathione (GSH) depleting agent buthionine sulfoximine (BSO) (100μM) for 28h, 48h, and 72h. We found that the AC50 (concentration that achieved 50% apoptosis) was 40μM for Ramos at 72h, 40μM for Granta at 48h and >80μM for L428 at 48h, while in the presence of BSO it was <10μM for Ramos at 72h, between 30–40μM for Granta at 28h and between 30–40μM for L428 at 48h. Apoptosis was completely blocked, by NAC, both in the presence and absence of BSO. Further, ANDRO induced PARP cleavage and activation of caspases 3, 8, and 9 in Granta and Ramos. Next, we explored the relationship of ANDRO and Forkhead transcription factors. ANDRO caused dephosphorylation of FOXO3a or FOXO1, in a dose- and time-dependent manner, and this was reversible by NAC. Downstream proteins of FOXO3a, Bim, p27kip1 and the isoforms of the autophagy-related protein LC3B were upregulated, and this was reversed by NAC. The LC3B isoform-II, which is cleaved from LC3B-I, is a marker of autophagy activation. To determine the role of autophagy in cell death related to ANDRO, we inhibited autophagy with 3-methyladenine (1–2mM) and found significant enhancement of ANDRO-induced apoptosis in Granta and Ramos. Finally, ANDRO induced apoptosis (>60% Annexin-V+/PI+) in malignant B-cells from a patient with chronic lymphocytic leukemia/small lymphocytic lymphoma (trisomy 12, peripheral blood absolute lymphocyte count 95.2 K/uL, bulky adenopathy) very low concentrations (5μM at 18h) in vitro, which was also reversible with NAC. We conclude that ANDRO induces ROS-dependent apoptosis in lymphoma cell lines and in a primary tumor sample, which is enhanced by depletion of GSH and inhibited by the antioxidant NAC. These effects appear to proceed through caspase activation and inhibition of autophagy, and are in part dependent on signaling through forkhead transcription factors and altered cellular redox pathways. Further studies of diterpenoids as single agents or in combination with other anti-lymphoma agents are warranted.

XAS Study of Arsenic Coordination in Euglena gracilis Exposed to Arsenite

Among the few eukaryotes adapted to the extreme conditions prevailing in acid mine drainage, Euglenae are ubiquitous in these metal(loid)-impacted environments, where they can be exposed to As(III) concentrations up to a few hundreds of mg x L(-1). In order to evaluate their resistance to this toxic metalloid and to identify associated detoxification mechanisms, we investigated arsenic coordination in the model photosynthetic protozoan, Euglena gracilis, cultured at pH 3.2 and exposed to As(III) at concentrations ranging from 10 to 500 mg x L(-1). E. gracilis is shown to tolerate As(III) concentrations up to 200 mg * L(-1), without accumulating this metalloid. X-ray absorption spectroscopy at the As K-edge shows that, in the cells, arsenic mainly binds to sulfur ligands, likely in the form of arsenic-trisglutathione (As-(GS)3) or arsenic-phytochelatin (As-PC) complexes, and to a much lesser extent to carbon ligands, presumably in the form of methylated As(III)-compounds. The key role of the glutathione pathway in As(III) detoxification is confirmed by the lower growth rate of E. gracilis cultures exposed to arsenic, in the presence of buthionine sulfoximine, an inhibitor of glutathione synthesis. This study provides the first investigation at the molecular scale of intracellular arsenic speciation in E. gracilis and thus contributes to the understanding of arsenic detoxification mechanisms in a eukaryotic microorganism under extreme acid mine drainage conditions.

Buthionine sulfoximine (BSO)-mediated improvement in cultured embryo quality in vitro entails changes in ascorbate metabolism, meristem development and embryo maturation

Applications of buthionine sulfoximine (BSO), an inhibitor of GSH (reduced glutathione), which switches the cellular glutathione pool towards the oxidized form GSSG, positively influences embryo quality by improving the structure of the shoot apical meristem and promoting embryo maturation, both of which improve the post-embryonic performance of the embryos. To investigate the mechanisms underlying BSO-mediated improvement in embryo quality the transcript profiles of developing Brassica napus microspore-derived embryos cultured in the absence (control) or presence of BSO were analyzed using a 15,000-element B. napus oligo microarray. BSO applications induced major changes in transcript accumulation patterns, especially during the late phases of embryogenesis. BSO affected the transcription and activities of key enzymes involved in ascorbate metabolism, which resulted in major fluctuations in cellular ascorbate levels. These changes were related to morphological characteristics of the embryos and their post-embryonic performance. BSO applications also activated many genes controlling meristem formation and function, including ZWILLE, SHOOTMERISTEMLESS, and ARGONAUTE 1. Increased expression of these genes may contribute to the improved structural quality of the shoot poles observed in the presence of BSO. Compared to their control counterparts, middle- and late-stage BSO-treated embryos also showed increased accumulation of transcripts associated with the maturation phase of zygotic embryo development, including genes encoding ABA-responsive proteins and storage- and late-embryogenic abundant (LEA) proteins. Overall these transcriptional changes support the observation that the BSO-induced oxidized glutathione redox state allows cultured embryos to reach both morphological and physiological maturity, which in turn guarantees successful regeneration and enhanced post-embryonic growth.

Bovine in vitro oocyte maturation as a model for manipulation of the γ-glutamyl cycle and intraoocyte glutathione

Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.

Constitutive histone H2AX phosphorylation on Ser-139 in cells untreated by genotoxic agents is cell-cycle phase specific and attenuated by scavenging reactive oxygen species

DNA damage, particularly when it involves formation of double-strand breaks (DSBs), triggers phosphorylation of histone H2AX on Ser-139. Phosphorylated H2AX has been named gammaH2AX, and induction of gammaH2AX in cells exposed to genotoxic agents is considered a sensitive and specific reporter of DNA damage. However, in untreated normal cells as well in the cells of various tumor lines cells, a fraction of histone H2AX molecules remain phosphorylated. In the present study, we observed that the extent of this constitutive H2AX phosphorylation varies depending on the cell type (line) and on cell cycle phase and, in most cell types, S and G(2)/M phase cells exhibit greater levels of H2AX phosphorylation than do cells in the G(1) phase. Furthermore, constitutive H2AX phosphorylation in human pulmonary carcinoma A549, lymphoblastoid TK6, and in normal bronchial epithelial cells was reduced following cell exposure to N-acetyl-L-cysteine, a scavenger of reactive oxygen intermediates; the reduction was most pronounced for G(2)M cells. Growth of A549 cells in the presence of buthionine sulfoximine, an inhibitor of glutathione synthetase, amplified the level of constitutive H2AX phosphorylation in A549 cells. The observed constitutive H2AX phosphorylation may be a reflection of the ongoing DNA damage mediated by reactive oxygen species (ROS) generated by metabolic activity during progression through the cell cycle, leading to formation of DSBs during the S phase. Because cumulative DNA damage in proliferating cells mediated by ROS is considered the key mechanism for cell ageing, the present approach to estimate the degree of attenuation of constitutive H2AX phosphorylation by antioxidants may provide a convenient tool to assess the DNA-protective and possible anti-ageing properties of other agents.

Improved development of microspore‐derived embryo cultures of Brassica napus cv Topaz following changes in glutathione metabolism

Glutathione has been shown to play an important role during embryo development in both plant and animal systems. The effects of altered glutathione metabolism during microspore‐derived embryos (MDEs) of Brassica napus were investigated following exogenous application of reduced glutathione (GSH), its oxidized form (GSSG) and buthionine sulfoximine (BSO), an inhibitor of glutathione de novo synthesis. Applications of BSO which lowered the cellular glutathione redox status, i.e. GSH/(GSH + GSSG), enhanced significantly the quality of the embryos and their ability to convert into viable plants. Histological analyses revealed that inclusions of BSO in the culture medium altered the pattern of storage product accumulation in the embryos and improved the architecture of the shoot apical meristems (SAMs). Compared with their control counterparts which showed severe signs of SAM deterioration, such as the formation of intercellular spaces and differentiation of the meristematic cells, BSO‐treated embryos had well‐organized SAMs. The improved SAM organization observed in the presence of BSO also correlated with the proper localization pattern of WUSCHEL, a SAM molecular marker gene which was miss‐expressed in control embryos. The beneficial effects of BSO on embryo development and conversion were ascribed to the increasing levels of ABA. The concentration of this growth regulator in BSO‐treated embryos was always higher than that of control embryos during the second half of the maturation period. Furthermore, many structural alterations induced by BSO could be reproduced in embryos cultured in the presence of ABA. Taken together, these results suggest that a lowering of the glutathione redox status during embryo development may represent a metabolic switch needed for increasing the endogenous levels of ABA, which is required for successful completion of the developmental program.