Reactive Oxidative Stress Levels Research Articles

Metabolomic analysis reveals the responses of docosahexaenoic-acid-producing Schizochytrium under hyposalinity conditions

The heterotrophic Schizochytrium sp. has been utilized to produce docosahexaenoic acids commercially in recent years. However, the high salinity required for Schizochytrium growth and docosahexaenoic acid production could result in salt-induced bioreactor corrosion and high downstream water treatment costs. Thus, exploring the physiological and metabolomic changes of Schizochytrium exposed to low-salt condition is beneficial for developing strategies of reducing the salt concentration during fermentation. In this study, Schizochytrium was exposed to 20, 10, 8, or 6 g/L of sea salt and the responses were determined. It was discovered that biomass accumulation was decreased, while starch and fatty acid was increased, especially unsaturated fatty acid accumulation in Schizochytrium, along with the salt concentration decrease. In addition, the metabolomic analysis showed that the intracellular pool sizes of NADPH, CoA, and ADP-GCS were significantly increased, while those of PEP and Ac-CoA were decreased, consistent with the content changes of fatty acid, starch, and biomass under hyposaline stress. The metabolomic analysis combined with further biochemical evidence revealed that fatty acid content elevation involved strengthening pentose phosphate pathway to provide more NADPH content; elevating CoA content to provide more precursors; upregulating inositol content, which was later demonstrated to enhance fatty acid accumulation; and activating the antioxidant system to reduce oxidation of unsaturated fatty acid caused by the reactive oxidative stress level. Finally, decreased content of glycerol, an important osmoregulator, along with enhanced cell wall polysaccharides, were also discovered in Schizochytrium as an adaptation to low-salt growth conditions. Our paper provides important guidelines for engineering Schizochytrium for low-salt cultivation in the future.

Leucine Supplementation in Middle-Aged Male Mice Improved Aging-Induced Vascular Remodeling and Dysfunction via Activating the Sirt1-Foxo1 Axis.

Vascular aging is associated with metabolic remodeling, and most studies focused on fatty acid and glucose metabolism. Based on our metabolomic data, leucine was significantly reduced in the aortas of aged mice. Whether leucine supplementation can reverse aging-induced vascular remodeling remains unknown. To investigate the effectiveness of leucine, male mice at 15 or 18 months were supplemented with leucine (1.5%) for 3 months. All the aged mice, with or without leucine, were sacrificed at 21 months. Blood pressure and vascular relaxation were measured. H&E, Masson’s trichrome, and Elastica van Gieson staining were used to assess aortic morphology. Vascular inflammation, reactive oxidative stress (ROS), and vascular smooth muscle cell (VSMC) phenotype were also measured in mouse aortas. Compared with the 21-month-old mice without leucine, leucine supplementation from 15 months significantly improved vascular relaxation, maintained the contractile phenotype of VSMCs, and repressed vascular inflammation and ROS levels. These benefits were not observed in the mice supplemented with leucine starting from 18 months, which was likely due to the reduction in leucine transporters Slc3a2 or Slc7a5 at 18 months. Furthermore, we found benefits from leucine via activating the Sirt1-induced Foxo1 deacetylation. Our findings indicated that leucine supplementation in middle-aged mice improved aging-induced vascular remodeling and dysfunction.

Toxic Effects and Mechanisms of Silver and Zinc Oxide Nanoparticles on Zebrafish Embryos in Aquatic Ecosystems.

The global application of engineered nanomaterials and nanoparticles (ENPs) in commercial products, industry, and medical fields has raised some concerns about their safety. These nanoparticles may gain access into rivers and marine environments through industrial or household wastewater discharge and thereby affect the ecosystem. In this study, we investigated the effects of silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnONPs) on zebrafish embryos in aquatic environments. We aimed to characterize the AgNP and ZnONP aggregates in natural waters, such as lakes, reservoirs, and rivers, and to determine whether they are toxic to developing zebrafish embryos. Different toxic effects and mechanisms were investigated by measuring the survival rate, hatching rate, body length, reactive oxidative stress (ROS) level, apoptosis, and autophagy. Spiking AgNPs or ZnONPs into natural water samples led to significant acute toxicity to zebrafish embryos, whereas the level of acute toxicity was relatively low when compared to Milli-Q (MQ) water, indicating the interaction and transformation of AgNPs or ZnONPs with complex components in a water environment that led to reduced toxicity. ZnONPs, but not AgNPs, triggered a significant delay of embryo hatching. Zebrafish embryos exposed to filtered natural water spiked with AgNPs or ZnONPs exhibited increased ROS levels, apoptosis, and lysosomal activity, an indicator of autophagy. Since autophagy is considered as an early indicator of ENP interactions with cells and has been recognized as an important mechanism of ENP-induced toxicity, developing a transgenic zebrafish system to detect ENP-induced autophagy may be an ideal strategy for predicting possible ecotoxicity that can be applied in the future for the risk assessment of ENPs.

Acacetin exerts antioxidant potential against atherosclerosis through Nrf2 pathway in apoE-/- Mice.

Oxidative stress has a considerable influence on endothelial cell dysfunction and atherosclerosis. Acacetin, an anti‐inflammatory and antiarrhythmic, is frequently used in the treatment of myocarditis, albeit its role in managing atherosclerosis is currently unclear. Thus, we evaluated the regulatory effects of acacetin in maintaining endothelial cell function and further investigated whether the flavonoid could attenuate atherosclerosis in apolipoprotein E deficiency (apoE−/−) mice. Different concentrations of acacetin were tested on EA.hy926 cells, either induced or non‐induced by human oxidized low‐density lipoprotein (oxLDL), to clarify its influence on cell viability, cellular reactive oxidative stress (ROS) level, apoptotic ratios and other regulatory effects. In vivo, apoE−/− mice were fed either a Western diet or a chow diet. Acacetin pro‐drug (15 mg/kg) was injected subcutaneously two times a day for 12 weeks. The effects of acacetin on the atherosclerotic process, plasma inflammatory factors and lipid metabolism were also investigated. Acacetin significantly increased EA.hy926 cell viability by reducing the ratios of apoptotic and necrotic cells at 3 μmol/L. Moreover, 3 μmol/L acacetin clearly decreased ROS levels and enhanced reductase protein expression through MsrA and Nrf2 pathway through phosphorylation of Nrf2 and degradation of Keap1. In vivo, acacetin treatment remarkably attenuated atherosclerosis by increasing reductase levels in circulation and aortic roots, decreasing plasma inflammatory factor levels as well as accelerating lipid metabolism in Western diet‐fed apoE−/− mice. Our findings demonstrate the anti‐oxidative and anti‐atherosclerotic effects of acacetin, in turn suggesting its potential therapeutic value in atherosclerotic‐related cardiovascular diseases (CVD).

Urban particulate matter regulates tight junction proteins by inducing oxidative stress via the Akt signal pathway in human nasal epithelial cells

Recent studies have revealed that increased reactive oxidative stress (ROS) induced by particulate matter (PM) affects tight junction (TJ) functions; however, the molecular mechanisms underlying this effect have not been evaluated fully. Cultured human epithelial cells obtained from inferior turbinate tissues were exposed to an urban PM (UPM) standard reference material (SRM 1648a). Intracellular ROS level and expression of proinflammatory cytokines and TJ proteins were examined. Expression level of phosphorylated (p)-Akt, p38, p65 were compared between exposed and unexposed cells. Cells were pretreated with the ROS scavenger N-acetylcysteine (NAC) or Akt inhibitor MK-2206 before exposure to determine whether the changes in cellular ROS and TJ protein expression could be reversed. Exposure to UPM significantly increased ROS levels and inflammatory cytokine expression levels, and decreased expression of TJ proteins zonula occludins (ZO)-1, occludin, claudin-1, and E-cadherin. UPM exposure increased p-Akt, p-p38, and p65 expression levels, and NAC pretreatment reversed these effects. Akt inhibition decreased UPM-induced ROS formation and p38 and p65 protein phosphorylation, and restored the decreased ZO-1 and E-cadherin expression. Akt inhibition and ROS scavenging may provide targets for maintaining epithelial integrity by restoring decreased TJ protein expression during exposure to UPM.

Abstract C60: Vitamin C inhibits the survival and growth of colorectal cancer cells with KRAS or BRAF mutations

Abstract Although more than half of colorectal cancer patients have either KRAS or BRAF mutations, targeted therapies for these subgroups of cancer patients are lacking. We previously showed that isogenic human colorectal cancer cell (CRC) lines harboring a KRAS or BRAF mutation up-regulate the expression of glucose transporter 1 (GLUT1), and increase glucose uptake and glycolysis. In this study, we go on to show that KRAS or BRAF mutant cells exhibit a significant increase in the uptake of vitamin C, mainly in its oxidized form, dehydroascorbic acid (DHA), relative to their wild-type counterparts through GLUT1. Notably, CRCs did not uptake vitamin C in its reduced form, possibly due to the fact that its known transporters, sodium vitamin C cotransporters (SVCTs) were generally not present in CRCs. Deletion of GLUT1 in mutant cells, overexpression of GLUT1 in wild-type cells and competition assays between glucose and DHA confirmed that GLUT1 is both necessary and sufficient for the uptake of vitamin C in CRCs. Both mutant and wild-type cells were able to grow in low glucose (1-2 mM) at a similar rate. In the same condition, we found vitamin C to be selectively toxic to KRAS and BRAF mutant cells, causing them to undergo apoptosis. In contrast, in high glucose levels (>10 mM), a similar concentration of vitamin C had no effect on the survival and growth of mutant cells. In addition, vitamin C significantly inhibited the growth of cancer cells with KRAS or BRAF mutations in vivo xenografts models. To find the mechanism by which vitamin C induces cell death in KRAS or BRAF mutant cells, we performed targeted metabolomics using LC/MS. Both unlabeled total metabolite analysis and flux analysis with C13-Glucose isotope strongly suggested that vitamin C re-routes glucose usage from aerobic glycolysis and non-oxidative pentose phosphate pathway (PPP) to the oxidative PPP to compensate for the lower levels of NADPH which was rapidly consumed to reduce GSSH to GSH. Having taken up exclusively DHA, the oxidized state of vitamin C, the mutant cells immediately use GSH to reduce Vitamin C, resulting in the accumulation of GSSH. Shunting glucose to the oxidative PPP reduces aerobic glycolysis, lowering ATP levels, and decreases glucose anabolism, disfavoring the synthesis of nucleic acids, lipid and amino acids, all needed for the survival and growth of mutant cells. Surprisingly the level of reactive oxidative stress (ROS) was largely unchanged following vitamin C treatment possibly because increased levels of vitamin C served as an anti-oxidant in mutant cells. There are currently more than three clinical trials phase I/II examining the effect of high dose vitamin C via intravenous administration in patients against lymphoma or solid cancers including CRCs. Our findings will provide proof of concept that KRAS or BRAF mutations in CRC can be predictive biomarkers for vitamin C therapy. Citation Format: Jihye Yun, Adam Kavalier, Jatin Roper, Steve Gross, Carlo Rago, Nickolas Papadopoulos, Bert Vogelstein, Lewis Cantley. Vitamin C inhibits the survival and growth of colorectal cancer cells with KRAS or BRAF mutations. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C60.

Activation of Oxidative Stress and Down-Regulation of Nuclear Factor Erythroid 2-Related Factor May Be Responsible for Disulfiram/Copper Complex Induced Apoptosis in Lymphoid Malignancy Cell Lines

Abstract 4869Disulfiram (DS), an antialcoholism drug, demonstrates strong antitumor activity in a copper (Cu)-dependent manner. Our previous study showed that it is highly cytotoxic in doxorubicin resistant leukemia cells and enhances cytotoxicity of doxorubicin. DS/Cu induces reactive oxidative stress (ROS) which activates stress related signaling pathway (c-Jun-amino-terminal kinase, JNK). Cancer cells possess higher ROS activity and some antiapoptotic factors. Therefore cancer cells may be effectively targeted by simultaneous inducing ROS and inhibiting antiapoptotic factors. Our study investigated the cytotoxicity of DS/Cu complex in acute lymphoblastic leukemia and Burkitt's lymphoma cell lines.MTT assay showed that at a low concentration (1μM) of Cu2+, DS induces cytotoxicity to Molt4 and Raji cells with IC50s of 0.435±0.109μM and 0.085±0.015μM respectively. The morphology and Annexin-V/PI flow cytometric analysis indicated that DSF/Cu induced apoptosis of Molt4 and Raji cells. The apoptotic proportion of Molt4 cells increased from 17.75% to 79.5% when exposed to increasing concentrations of DS (0.125, 0.25, 0.5, 1μM) for 24h. The DS/Cu induced apoptosis is also time-dependent. The apoptotic proportion of Raji cells increased from 18.89±5.86% to 81.03±7.91% when exposed to DS (3.3μM) and Cu (1μM) for 6, 12 and 24h. Nrf2 is a key antioxidant factor. Western blot indicated that Nrf2 nuclear translocation was changed in a time-dependent manner after cells being treated by DS/Cu. Nrf2 expression was increased when Raji cells were treated for less than 12h and decreased after 18h or 24h treatment (Figure 1). Inhibition of Nrf2 could also be seen in Molt4 cells after 24h treatment. QT-PCR showed that DS/Cu down-regulated Nrf2 gene expression in a concentration dependent manner. ROS levels are closely related to Nrf2. Flow cytometric analysis showed that DS/Cu induced ROS generation. Western blot manifested that DS/Cu complex induced phosphorylation of JNK expression and inhibited P65 activity (Figure 2). N-acetyl-L-cysteine (NAC), an antioxidant, can partially attenuate DS/Cu complex-induced apoptosis, restore Nrf2 nuclear translocation, reactivate P65 activity and block JNK activation (Figure 3).In conclusion, our study showed that DS/Cu induces apoptosis in vitro and shows anticancer activity in vivo to lymphoid malignancy cell lines. We also demonstrated that ROS played a key role in DS/Cu- induced apoptosis. ROS can partially inhibit P65 activity and activate JNK while having bi phase regulation of Nrf2 expression. The initially increased Nrf2 expression decreases after 18h and 24h treatment. Therefore the DS/Cu induced ROS may be higher than that antioxidant factors could protect and thus the Nrf2-mediated cellular survival mechanism was disabled through down regulation of Nrf2 to allow initiation of death process. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.