Presence Of N-acetyl-L-cysteine Research Articles

N-Acetyl-l-cysteine-Loaded Nanosystems as a Promising Therapeutic Approach Toward the Eradication of Pseudomonas aeruginosa Biofilms.

Bacterial biofilms are a major health concern, mainly due to their contribution to increased bacterial resistance to well-known antibiotics. The conventional treatment of biofilms represents a challenge, and frequently, eradication is not achieved with long-lasting administration of antibiotics. In this context, the present work proposes an innovative therapeutic approach that is focused on the encapsulation of N-acetyl-l-cysteine (NAC) into lipid nanoparticles (LNPs) functionalized with d-amino acids to target and disrupt bacterial biofilms. The optimized formulations presented a mean hydrodynamic diameter around 200 nm, a low polydispersity index, and a high loading capacity. These formulations were stable under storage conditions up to 6 months. In vitro biocompatibility studies showed a low cytotoxicity effect in fibroblasts and a low hemolytic activity in human red blood cells. Nevertheless, unloaded LNPs showed a higher hemolytic potential than NAC-loaded LNPs, which suggests a safer profile of the latter. The in vitro antibiofilm efficacy of the developed formulations was tested against Staphylococcus epidermidis (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) mature biofilms. The results showed that the NAC-loaded LNPs were ineffective against S. epidermidis biofilms, while a significant reduction of biofilm biomass and bacterial viability in P. aeruginosa biofilms were observed. In a more complex therapeutic approach, the LNPs were further combined with moxifloxacin, revealing a beneficial effect between the LNPs and the antibiotic against P. aeruginosa biofilms. Both alone and in combination with moxifloxacin, unloaded and NAC-loaded LNPs functionalized with d-amino acids showed a great potential to reduce bacterial viability, with no significant differences in the presence or absence of NAC. However, the presence of NAC in NAC-loaded functionalized LNPs shows a safer profile than the unloaded LNPs, which is beneficial for an in vivo application. Overall, the developed formulations present a potential therapeutic approach against P. aeruginosa biofilms, alone or in combination with antibiotics.

Synthesis, characterization, and cytotoxicity assessment of N-acetyl-l-cysteine capped ZnO nanoparticles as camptothecin delivery system.

The chemical stability, good biocompatibility and high drug loading capacity of zinc oxide nanoparticles (ZnO NPs) and their biomedical potentials make them a promising candidate for drug delivery. The aim of this study was to develop and assess a simple procedure for surface functionalization of ZnO NPs by N-acetyl-l-cysteine (NAC) for anticancer camptothecin (CPT) delivery. NAC capped ZnO NPs were successfully made using ZnCl2 and NaOH in the presence of NAC. CPT was covalently conjugated to the surface of as-synthesized ZnO-NAC NPs. To characterize the synthesized conjugate product (ZnO-NAC-CPT NPs), X-ray diffraction, Fourier Transform Infrared spectroscopy, transmission electron microscopy, scanning electron microscopy, and dynamic light scattering method were used. Our results indicated that the ZnO-NAC-CPT NPs exhibit near-spherical morphology and uniform dispersion with an average diameter of ∼70 nm. The hemolysis assay showed that ZnO-NAC-CPT NPs has almost no hemolytic activity. In addition, MTT cytotoxicity assessment on A549 lung cancer cells revealed a drop of IC50 values from 1.17 μg/mL (free CPT) to 0.66 μg/mL (ZnO-NAC-CPT NPs). This result showed an augmented cancer-inhibitory effect of nanoconjugate complex. In conclusion, the novel ZnO-NAC-CPT NPs could be considered for new therapeutic endeavors.

Piceatannol-Induced Apoptosis Is Reversed by N-Acetyl-L-cysteine through Restoration of XIAP Expression.

Piceatannol, a naturally occurring stilbene derivative mainly found in grapes, possesses apoptotic activity in various cancer cell lines, in addition to potent antioxidant activity. In the current study, we showed that piceatannol exhibits potent cytotoxic effects in all tested leukemia cell lines (THP-1, HL-60, U937, and K562). These effects were accompanied by induction of DNA damage, an increase in the proportion of cells in the sub-G1 phase of the cell cycle, and inhibition of reactive oxygen species (ROS) generation. However, N-acetyl-L-cysteine (NAC), a strong ROS scavenger, significantly inhibited piceatannol-induced apoptosis, suggesting that piceatannol-induced apoptosis does not occur via inhibition of ROS generation. Piceatannol also resulted in a significant increase in mitochondrial depolarization, along with a decline in Bcl-2 expression, which was not restored by NAC. Conversely, ectopic Bcl-2 overexpression moderately inhibited piceatannol-induced apoptosis. Furthermore, piceatannol strongly inhibited X-linked inhibitor of apoptosis protein (XIAP) expression, which was restored by NAC. A transient knockdown of XIAP significantly increased piceatannol-induced apoptosis in the presence of NAC, suggesting that XIAP downregulation increases piceatannol-induced apoptosis, and that NAC could reverse this effect by increasing XIAP expression. Taken together, these results suggest that piceatannol induces apoptosis in human leukemia cell lines by downregulating XIAP expression, regardless of antioxidant activity.

Therapeutic effects of N-acetyl-L-cysteine on liver damage induced by long-term CCl4 administration.

N-acetyl-L-cysteine (NAC) is a drug routinely used in several health problems, e.g. liver damage. There is some information emerged on its negative effects in certain situations. The aim of our study was to examine its ability to influence liver damage induced by long-term burden. We induced liver damage by CCl4 (10 weeks) and monitored the impact of parallel NAC administration (daily 150 mg/kg of b.w.) on liver morphology and some biochemical parameters (triacylglycerols, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, bile acids, proteins, albumins and cholinesterase). NAC significantly decreased levels of bile acids and bilirubin in plasma and triacylglycerols in liver, all of them elevated by impairment with CCl4. Reduction of cholesterol induced by CCl4 was completely recovered in the presence of NAC as indicated by its elevation to control levels. NAC administration did not improve the histological parameters. Together with protective effects of NAC, we found also its deleterious properties: parallel administration of CCl4 and NAC increased triacylglycerols, ALT and AST activity and significantly increased plasma cholinesterase activity. We have observed nonsignificantly increased percentage of liver tissue fibrosis. Our results have shown that NAC administered simultaneously with liver damaging agent CCl4, exhibits not only protective, but also deleterious effects as indicated by several biochemical parameters.

Triclosan activates aryl hydrocarbon receptor (AhR)-dependent apoptosis and affects Cyp1a1 and Cyp1b1 expression in mouse neocortical neurons

Triclosan (TCS) is an antimicrobial agent that is used extensively in personal care and in sanitizing products, such as soaps, toothpastes, and hair products. A number of studies have revealed the presence of TCS in human tissues, such as fat, liver and brain, in addition to blood and breast milk. The aim of the present study was to investigate the impact of TCS on AhR and Cyp1a1/Cyp1b1 signaling in mouse neocortical neurons in primary cultures. In addition to the use of selective ligands and siRNAs, expression levels of mRNA and proteins as well as caspase-3 activity, reactive oxygen species (ROS) formation, and lactate dehydrogenase (LDH) release have been measured. We also studied the involvement of the AhR in TCS-induced LDH release and caspase-3 activation as well as the effect of TCS on ROS generation. Cultures of neocortical neurons were prepared from Swiss mouse embryos on day 15/16 of gestation. The cells were cultured in phenol red-free Neurobasal medium with B27 and glutamine, and the neurons were exposed to 1 and 10µM TCS. Our experiments showed that the expression of AhR and Cyp1a1 mRNA decreased in cells exposed to 10µM TCS for 3 or 6h. In the case of Cyp1b1, mRNA expression remained unchanged compared with the control group following 3h of exposure to TCS, but after 6h, the mRNA expression of Cyp1b1 was decreased. Our results confirmed that the AhR is involved in the TCS mechanism of action, and our data demonstrated that after the cells were transfected with AhR siRNA, the cytotoxic and pro-apoptotic properties of TCS were decreased. The decrease in Cyp1a1 mRNA and protein expression levels accompanied by a decrease in its activity. The stimulation of Cyp1a1 activity produced by the application of an AhR agonist (βNF) was attenuated by TCS, whereas the addition of AhR antagonist (αNF) reversed the inhibitory effects of TCS. In our experiments, TCS diminished Cyp1b1 mRNA and enhanced its protein expression. In case of Cyp1a1 we observed paradoxical effect of TCS action, which caused the decrease in activity and protein expression of Cyp1a1 and the increase in protein level of AhR. Therefore, we determined the effects of TCS on the production of ROS. Our results revealed that TCS increased the production of ROS and that this effect of TCS was reversed by 10µM N-acetyl-L-cysteine (NAC), the ROS scavenger. To confirm an involvement of ROS in TCS-induced neurotoxicity we measured AhR, Cyp1a1, and Cyp1b1 mRNA expression levels in cells co-treated with TCS and NAC. In the presence of NAC, TCS enhanced mRNA expression of the cytochromes and AhR at 3 and 6h, respectively. We postulate that TCS exhibits primary and secondary effects. The primary effects such as impairment of Cyp1a1 signaling are mediated by TCS-induced ROS production, whereas secondary effects of TCS are due to transcriptional activity of AhR and estrogenic properties of TCS.

Abstract 5046: High-throughput chemical screening for sensitization of bladder cancer to gemcitabine and cisplatin chemotherapy

Abstract Background Gemcitabine and cisplatin chemotherapy (GC) is the current standard regimen for locally advanced and metastatic bladder cancer (BC). Despite a relatively high initial response rate, some cases do not regress (intrinsic resistance) and the remaining cases often show regrowth after initial shrinkage (acquired resistance). To identify novel therapeutic agents for overcoming these resistances, we applied a high-throughput screening of chemicals administered in combination with GC. Methods and Findings As a high-throughput screening, 2100 compounds were administered alone or in combination with GC to human BC cell lines (J82, UMUC-3). Cell viability was determined after 3-day incubation and chemicals that enhanced inhibitory effect of GC were screened. The initial screening identified 26 compounds and further validation narrowed them into the most synergistic agent disulfiram (DSF), an FDA-approved drug for alcoholism. Combination index assay showed synergistic effects of DSF with cisplatin but not with gemcitabine in J82, UMUC-3, T24, HT1197 and HT1376 cells. Co-administration of DSF significantly enhanced apoptosis by GC treatment in UMUC-3 and T24 cells, with significant increase of reactive oxygen species (ROS) production. DSF-induced apoptosis was attenuated in the presence of N-acetyl L-cysteine and Trolox, ROS scavengers. Use of DSF in combination with GC (GCD) significantly inhibited tumor growth of UMUC-3 subcutaneous xenograft on athymic mice (by 39% compared with GC alone, p = 0.02). GCD regimen was as tolerable as GC and no significant differences were observed in body weight of treated mice between the two regimen. Conclusion Repositioning of DSF to a chemotherapy sensitizer is a promising treatment strategy, which can be translated rapidly in the future. Citation Format: Yuki Kita, Takashi Kobayashi, Norihiko Masuda, Atsuro Sawada, Yoshiyuki Matsui, Takahiro Inoue, Tomomi Kamba, Osamu Ogawa. High-throughput chemical screening for sensitization of bladder cancer to gemcitabine and cisplatin chemotherapy. [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 5046.

Tetrabromobisphenol A (TBBPA)-stimulated reactive oxygen species (ROS) production in cell-free model using the 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) assay-limitations of method.

Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant, applied in a variety of commercial and household products, mainly electronic ones. Since the production of reactive oxygen species (ROS) is considered one of the principal cytotoxicity mechanisms, numerous studies undertake that aspect of TBBPA’s mechanism of action. The present study verifies if the fluorogenic substrate 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) should be used to detect ROS production induced by TBBPA. To determine the ability of TBBPA alone to stimulate the conversion of H2DCFDA to its fluorescent product 2’,7’-dichlorofluorescein (DCF), we used a cell-free model. In the experiments we check different cultured media also in combination with free radical scavenger N-acetyl-l-cysteine (NAC). Additionally, experiments with stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH·) have been made. The presented data showed that TBBPA in all tested concentrations interacts with H2DCFDA in phosphate-buffered saline (PBS) buffer while in micromolar concentrations in the DMEM/F12 medium with and without serum. The addition of NAC inhibited the interaction of TBBPA with H2DCFDA. Experiments with DPPH· showed that, in the presence of NAC, TBBPA acts like a free radical. TBBPA has similar properties to free radical and is susceptible to free radical scavenging properties of NAC. Our results indicated that H2DCFDA assay cannot be used to evaluate cellular ROS production in TBBPA studies. The study connected with TBBPA-stimulated ROS production in cell culture models using the H2DCFDA assay should be revised using a different method. However, due to the free radical-like nature of TBBPA, it can be very difficult. Therefore, further investigation of the nature of TBBPA as a compound with similar properties to free radical is required.

Abstract 3306: The RNA-binding protein HuR radiosensitizes human TNBC cells by modulating the cellular response to DNA damage and oxidative stress

Abstract The RNA-binding protein human antigen R (HuR) associates with U-/AU-rich mRNAs encoding proteins that control cell proliferation, metabolism and the stress response. HuR is overexpressed in several human cancers and its overexpression is associated with poor prognosis and resistance to therapy. While the role of HuR in drug resistance has been studied, its contribution to radiation resistance has not been examined. Therefore, we investigated the role of HuR in radiation resistance of triple negative breast cancer (TNBC) cells: MDA-MB-231, MDA-MB-468 and Hs578t. Reduction of HuR expression using small interfering (si) RNA decreased cell proliferation and sensitized TNBC cells to ionizing radiation. Clonogenic assays indicated that silencing HuR suppressed the clonogenic survival of all three TNBC cell lines with survival at 2 Gy (SF2) reduced from 59%, 49%, 65% in control cells to 40%, 33%, and 46% in siHuR-treated MDA-MB-231, MDA-MB-468 and Hs578t cells, respectively. To delineate the underlying mechanism of radiosensitization and to identify candidate mRNAs showing altered levels after silencing HuR, we undertook a ribonomic approach. First, since ionizing radiation enhances the production of reactive oxygen species (ROS), causing DNA damage, we investigated the possible involvement of ROS in siHuR-mediated radiosensitization. ROS production in control or HuR-silenced cells treated with or without radiation was measured using the fluorescent dye 2′-7′-Dichlorodihydrofluorescein diacetate (DCFDA). Radiation significantly increased ROS generation in HuR knockdown cells compared to control cells. To further test the involvement of ROS in radiosensitivity, control and HuR-silenced cells were pre-treated with N-Acetyl-L- cysteine (NAC), an ROS scavenger, prior to radiation. The presence of NAC completely prevented radiation sensitivity and ROS production, indicating the involvement of ROS in HuR-mediated radiation sensitivity. Second, we directly tested the involvement of the DNA damage response (DDR) pathway in radiosensitivity after silencing HuR by evaluating the number of γ-H2AX foci (a common indicator of DNA damage) in control and HuR-silenced cells following irradiation. Our results showed that the number of γ-H2AX foci was significantly greater in HuR-silenced cells than in control cells at 1 h, 2 h and 24 h after irradiation. The persistence of γ-H2AX foci suggests that radiosensitization by HuR silencing involves inhibition of the repair of damaged DNA. This hypothesis was supported by the comet assay, which showed that HuR-silenced cells had larger and longer-lasting tails than control cells, in keeping with the higher levels of DNA damage seen after silencing HuR. Our studies indicate that radiosensitization upon HuR knockdown is linked to suppression of the cellular response to genotoxic and oxidative damage. Citation Format: Meghna Mehta, James Griffith, Kanthesh Basalingappa, Anish Babu, Narsireddy Amreddy, Ranganayaki Muralidharan, Myriam Gorospe, Terence Herman, Wei-Qun Ding, Rajagopal Ramesh, Anupama Munshi. The RNA-binding protein HuR radiosensitizes human TNBC cells by modulating the cellular response to DNA damage and oxidative stress. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3306. doi:10.1158/1538-7445.AM2015-3306

Involvement of Hydrogen Peroxide in Safingol-Induced Endonuclease G-Mediated Apoptosis of Squamous Cell Carcinoma Cells

Safingol, a L-threo-dihydrosphingosine, induced the nuclear translocation of a mitochondrial apoptogenic mediator—endonuclease G (endo G)—and apoptosis of human oral squamous cell carcinoma (SCC) cells. Upstream mediators remain largely unknown. The levels of hydrogen peroxide (H2O2) in cultured oral SCC cells were measured. Treatment with safingol increased intracellular H2O2 levels but not extracellular H2O2 levels, indicating the production of H2O2. The cell killing effect of safingol and H2O2 was diminished in the presence of reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC). Dual staining of cells with annexin V and propidium iodide (PI) revealed that apoptotic cell death occurred by treatment with H2O2 and safingol. The number of apoptotic cells was reduced in the presence of NAC. In untreated cells, endo G distributed in the cytoplasm and an association of endo G with mitochondria was observed. After treatment with H2O2 and safingol, endo G was distributed to the nucleus and cytoplasm, indicating the nuclear translocation of the mitochondrial factor. NAC prevented the increase of apoptotic cells and the translocation of endo G. Knock down of endo G diminished the cell killing effect of H2O2 and safingol. These results suggest that H2O2 is involved in the endo G-mediated apoptosis of oral SCC cells by safingol.

Superoxide Mediates Direct Current Electric Field-Induced Directional Migration of Glioma Cells through the Activation of AKT and ERK

Direct current electric fields (DCEFs) can induce directional migration for many cell types through activation of intracellular signaling pathways. However, the mechanisms that bridge extracellular electrical stimulation with intracellular signaling remain largely unknown. In the current study, we found that a DCEF can induce the directional migration of U87, C6 and U251 glioma cells to the cathode and stimulate the production of hydrogen peroxide and superoxide. Subsequent studies demonstrated that the electrotaxis of glioma cells were abolished by the superoxide inhibitor N-acetyl-l-cysteine (NAC) or overexpression of mitochondrial superoxide dismutase (MnSOD), but was not affected by inhibition of hydrogen peroxide through the overexpression of catalase. Furthermore, we found that the presence of NAC, as well as the overexpression of MnSOD, could almost completely abolish the activation of Akt, extracellular-signal-regulated kinase (Erk)1/2, c-Jun N-terminal kinase (JNK), and p38, although only JNK and p38 were affected by overexpression of catalase. The presenting of specific inhibitors can decrease the activation of Erk1/2 or Akt as well as the directional migration of glioma cells. Collectively, our data demonstrate that superoxide may play a critical role in DCEF-induced directional migration of glioma cells through the regulation of Akt and Erk1/2 activation. This study provides novel evidence that the superoxide is at least one of the “bridges” coupling the extracellular electric stimulation to the intracellular signals during DCEF-mediated cell directional migration.

Role of oxygen free radicals in the proliferation of myofibroblasts induced by AngII

Previous studies have demonstrated the important role of angiotension II (AngII) in promoting proliferation of myofibroblasts (myoFbs) and myocardial fibrosis. However, the underlying mechanisms and the role of oxygen free radicals in the proliferation of myofibroblasts induced by AngII are unclear. The present study was designed to shed light on this issue through exploration of AngII signaling pathways via in vitro experiments. Primary cultures of neonatal rat myoFbs were divided into five groups which were treated with AngII (10−8 to 10−6M), AngII with the antioxidant N-acetyl-L-cysteine (NAC), or normal culture medium. We observed the proliferation of myoFbs as induced by AngII at different concentrations with MTT. Reactive oxygen species (ROS) levels in myoFbs were detected by monitoring the fluorescence of 2′,7′-dichlorofluorescein. The contents and levels of oxygen free radicals (OH) in the three groups were detected by spectrophotometer, immunocytochemical staining, and confocal fluorescence. Western blot and image analysis were used to measure membrane translocation and expression of phospho-protein kinase Cα. MyoFbs incubated with AngII (10−8 to 10−6M) for 24h increased their rate of proliferation, the content of OH, and expression of ROS (P<0.01 vs. control group), whereas these parameters decreased in the presence of NAC. Immunocytochemistry, confocal fluorescence staining and image analysis showed that AngII could promote the translocation and expression of p-PKCα in membrane, and the antioxidant NAC blocked this increase (P<0.01). Western blot results also showed that NAC could inhibit the expression of p-PKCα.

Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation.

Autophagy is an evolutionarily conserved catabolic process that maintains cellular homeostasis under stress conditions such as starvation and pathogen infection. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that plays important roles in inflammation and tumorigenesis. Cytokines such as IL-1β and TNF-α that are induced by MIF have been shown to be involved in the induction of autophagy. However, the actual role of MIF in autophagy remains unclear. Here, we have demonstrated that incubation of human hepatoma cell line HuH-7 cells with recombinant MIF (rMIF) induced reactive oxygen species (ROS) production and autophagy formation, including LC3-II expression, LC3 punctae formation, autophagic flux, and mitochondria membrane potential loss. The autophagy induced by rMIF was inhibited in the presence of MIF inhibitor, ISO-1 as well as ROS scavenger N-acetyl-L-cysteine (NAC). In addition, serum starvation-induced MIF release and autophagy of HuH-7 cells were partly blocked in the presence of NAC. Moreover, diminished MIF expression by shRNA transfection or inhibition of MIF by ISO-1 decreased serum starvation-induced autophagy of HuH-7 cells. Taken together, these data suggest that cell autophagy was induced by MIF under stress conditions such as inflammation and starvation through ROS generation.

Formation of the Thiol Conjugates and Active Metabolite of Clopidogrel by Human Liver Microsomes

We reported previously the formation of a glutathionyl conjugate of the active metabolite (AM) of clopidogrel and the covalent modification of a cysteinyl residue of human cytochrome P450 2B6 in a reconstituted system (Mol Pharmacol 80:839-847, 2011). In this work, we extended our studies of the metabolism of clopidogrel to human liver microsomes in the presence of four reductants, namely, GSH, l-Cys, N-acetyl-l-cysteine (NAC), and ascorbic acid. Our results demonstrated that formation of the AM was greatly affected by the reductant used and the relative amounts of the AM formed were increased in the following order: NAC (17%) < l-Cys (53%) < ascorbic acid (61%) < GSH (100%). AM-thiol conjugates were observed in the presence of NAC, l-Cys, and GSH. In the case of GSH, the formation of both the AM and the glutathionyl conjugate was dependent on the GSH concentrations, with similar K(m) values of ~0.5 mM, which indicates that formation of the thiol conjugates constitutes an integral part of the bioactivation processes for clopidogrel. It was observed that the AM was slowly converted to the thiol conjugate, with a half-life of ~10 h. Addition of dithiothreitol to the reaction mixture reversed the conversion, which resulted in a decrease in AM-thiol conjugate levels and a concomitant increase in AM levels, whereas addition of NAC led to the formation of AM-NAC and a concomitant decrease in AM-GSH levels. These results not only confirm that the AM is formed through oxidative opening of the thiolactone ring but also suggest the existence of an equilibrium between the AM, the thiol conjugates, and the reductants. These factors may affect the effective concentrations of the AM in vivo.

Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation (174.10)

Abstract Autophagy is an evolutionarily conserved catabolic process which maintains cellular homeostasis under stress conditions such as starvation and pathogen infection. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that plays important roles in inflammation and tumorigenesis. Cytokines such as IL-1β and TNF-α that are induced by MIF have been shown to be involved in the induction of autophagy. However, the actually role of MIF in autophagy is still unclear. Here, we demonstrated that incubation of human hepatoma cell line Huh-7 cells with recombinant MIF (rMIF) induced reactive oxygen species (ROS) production and autophagy formation, including LC3-II expression, LC3 punctae formation and mitochondria membrane potential loss. The autophagy induced by rMIF was inhibited in the presence of MIF inhibitor, ISO-1 as well as ROS scavenger N-acetyl-L-cysteine (NAC). In addition, serum starvation-induced MIF release and autophagy of Huh-7 cells were partly blocked in the presence of NAC. Moreover, diminished MIF expression by shRNA transfection or inhibition of MIF by ISO-1 decreased serum starvation-induced autophagy of Huh-7 cells. Taken together, these data suggest that MIF is involved in autophagy through ROS generation to prevent cell death.

Involvement of oxidative stress in taxol-induced apoptosis in chronic myelogenous leukemia K562 cells

It is now well accepted that taxol exhibits cytotoxicity and antitumor activity in many human tumors through microtubule stabilization and induction of G2/M cell cycle arrest with final extensive cell apoptosis. Since many anti-cancer agents exert their cytotoxic effects through reactive oxygen species (ROS), we were interested to evaluate whether oxidative stress is involved in taxol-induced cytotoxicity among human leukemia K562 cells. Our results showed that induction of apoptosis was associated with generation of ROS and glutathione (GSH) depletion. The increase in ROS production and apoptosis were both suppressed by antioxidant N-acetyl-l-cysteine (NAC). Moreover, taxol caused an increase in c-Jun NH(2)-terminal kinase (JNK) and p38 activities, two of the well known mediators of the stress activation pathways. Attenuation of JNK expression in the presence of NAC might indicate the modulation of the level of JNK activity by ROS. Furthermore, our data indicated that Bcl-2α was down-regulated in taxol-treated cells and its expression was modulated by ROS and JNK activity. The activities of caspase-9 and -3 were also increased upon treatment with taxol; however, pre-treatment of cells with NAC or JNK inhibitor (SP600125) impeded taxol-mediated caspase activation and apoptosis in K562 cells, suggesting that JNK acts upstream of the caspases. Taken together, these results indicate that taxol induces apoptosis in chronic myelogenous leukemia cells by inducing intracellular oxidative stress and JNK activation pathway.

Effect of N-acetyl-L-cysteine Supplementation in Semen Extenders on Semen Quality and Reactive Oxygen Species of Chilled Canine Spermatozoa

The objective of this study was to evaluate the quality of chilled dog semen processed with extenders containing various concentrations of N-acetyl-L-cysteine (NAC). Ejaculates from five dogs were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF-movement), viability, acrosomal integrity and by the hypo-osmotic swelling test (HOST). In addition, superoxide anion (O(2)(-*)) production, hydroxyl radicals (OH(*)) and total reactive oxygen species (tROS) were determined. The pool was divided into five aliquots, which were diluted to a final concentration of 66.66 x 10(6) spermatozoa/ml with Tris-glucose-egg yolk extender containing one of the following concentrations of NAC (0, 0.5, 1, 2.5 or 5 mm). The semen aliquots were chilled and preserved at 4 degrees C. Semen quality was evaluated after rewarming at 72 h. Sperm motility was significantly higher with the 0.5 mm concentration compared with the control group (p = 0.001). Rapid steady forward movement was higher with the 0.5 and 1 mm concentrations compared with the control and 5 mm group (p < 0.001). Viability and HOST percentages were not significantly altered. Compared with the control, the 5 mm concentration showed significantly reduced percentages of spermatozoa with normal acrosomes (p = 0.049). None of the ROS values at 72 h were significantly affected by the presence of NAC in semen extenders, although all NAC concentrations showed lower O(2)(-*) and OH(*) values compared with the control. Only the concentrations of 1 and 5 mm inhibited the significant increase of tROS values after 72 h, compared with the fresh semen value. In conclusion, NAC supplementation of semen extenders is beneficial to semen motility of canine spermatozoa during chilling with the 0.5 mm concentration being the most effective, although no significant ROS inhibition was observed at 72 h.

Formation of Three N-Acetyl-l-cysteine Monoadducts and One Diadduct by the Reaction of S-(1,2-Dichlorovinyl)-l-cysteine Sulfoxide with N-Acetyl-l-cysteine at Physiological Conditions: Chemical Mechanisms and Toxicological Implications

Previously, our laboratory has shown that S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS), a Michael acceptor produced by a flavin-containing monooxygenase 3 (FMO3)-mediated oxidation of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), is a more potent nephrotoxicant than DCVC. In the present study, we characterized reactions of DCVCS with nucleophilic amino acids. DCVCS incubations with N-acetyl-L-cysteine (NAC) at pH 7.4 and 37 degrees C for 1 h resulted in the formation of three monoadducts and one diadduct characterized by LC/MS, 1H NMR, and 1H-detected heteronuclear single quantum correlation. The formation of all adducts (with relative ratios of 29, 31, 24, and 12%, respectively) was rapid and time-dependent; the half-lives of the two DCVCS diastereomers in the presence of NAC were 13.8 (diastereomer I) and 9.4 min (diastereomer II). Adducts 1 and 2 were determined to be diastereomers of S-[1-chloro-2-(N-acetyl-L-cystein- S-yl)vinyl]-L-cysteine sulfoxide formed by Michael addition of NAC to the terminal vinylic carbon of DCVCS followed by loss of HCl. Adduct 4 was determined to be S-[2-chloro-2-(N-acetyl-L-cystein- S-yl)vinyl]-L-cysteine sulfoxide formed from the initial Michael addition product followed by a less favorable loss of HCl and/or by a rearrangement of adduct 2 through the formation of a cyclic chloronium ion. The addition of another molecule of NAC to monoadducts 1, 2, or 4 resulted in the formation of the novel diadduct, S-[2,2-( N-acetyl-L-cystein-S-yl)vinyl]-L-cysteine sulfoxide (adduct 3), whose detection in relatively large amount suggests that DCVCS could act as a cross-linking agent. DCVCS was not reactive with N-acetyl-L-lysine or L-valinamide at similar incubation conditions. Collectively, the results suggest selective reactivity of DCVCS toward protein sulfhydryl groups. Furthermore, the cross-linking properties of DCVCS may in part explain its high nephrotoxic potency.

Cannabidiol-induced apoptosis in primary lymphocytes is associated with oxidative stress-dependent activation of caspase-8

We recently reported that cannabidiol (CBD) exhibited a generalized suppressive effect on T-cell functional activities in splenocytes directly exposed to CBD in vitro or isolated from CBD-administered mice. To investigate the potential mechanisms of CBD effects on T cells, we characterized the pro-apoptotic effect of CBD on primary lymphocytes. The apoptosis of splenocytes was markedly enhanced following CBD exposure in a time- and concentration-dependent manner, as evidenced by nuclear hypodiploidity and DNA strand breaks. Exposure of splenocytes to CBD elicited an early production of reactive oxygen species (ROS) with the peak response at 1 h post CBD treatment. In parallel with the ROS production, a gradual diminishment in the cellular glutathione (GSH) content was detected in CBD-treated splenocytes. Both CBD-mediated ROS production and GSH diminishment were remarkably attenuated by the presence of N-acetyl- l-cysteine (NAC), a thiol antioxidant. In addition, CBD treatment significantly stimulated the activation of caspase-8, which was abrogated in the presence of NAC or GSH. Pretreatment of splenocytes with a cell-permeable inhibitor for caspase-8 significantly attenuated, in a concentration-dependent manner, CBD-mediated apoptosis, but not ROS production. Collectively, the present study demonstrated that the apoptotic effect of CBD in primary lymphocytes is closely associated with oxidative stress-dependent activation of caspase-8.

Antioxidant N‐acetyl‐l‐cysteine inhibits erythropoietin‐induced differentiation of erythroid progenitors derived from mouse fetal liver

To determine the role of reactive oxygen species in erythroid differentiation, we investigated the effects of an antioxidant, N-acetyl-L-cysteine (NAC), on the differentiation of erythroid progenitors derived from mouse fetal liver. In response to erythropoietin (Epo), erythroid progenitors undergo differentiation in vitro and express erythroid-specific genes such as betamajor-globin, Alas2, MafK, p45, Eklf, and Gata1. Expression of these genes was decreased in the presence of NAC, whereas the expression of c-myb, which is downregulated during erythroid differentiation, remained constant. Moreover, NAC treatment inhibited an increase in the number of cells expressing high levels of erythroid-specific antigen TER119. Treatment with another antioxidant, pyrrolidine dithiocarbamate, also caused the attenuation of TER119 expression. These results suggest that reactive oxygen species are involved in Epo-mediated erythroid differentiation.

Areca nut extract suppresses T-cell activation and interferon-γ production via the induction of oxidative stress

Areca quid chewing is a major risk factor associated with oral submucous fibrosis (OSF) and oral cancer. Experimental evidence indicates that immune deterioration is associated with the pathophysiology of OSF and oral cancer. In addition, reactive oxygen species (ROS) is shown to play a role in the cytotoxic and genotoxic effect induced by areca nut extracts (ANE) in oral cells. The present studies investigated the effects of ANE on T-cell reactivity and the role of ROS in ANE effects. Treatment of splenocytes with ANE induced a marked cytotoxic effect, and suppressed the production of IL-2 and IFN-γ, whereas the production of IL-4 was unaffected. The ANE-mediated cytotoxicity, and suppression of IFN-γ and IL-2 production were attenuated by the presence of antioxidant N-acetyl- l-cysteine (NAC). Moreover, flow cytometric analysis demonstrated an increase in cellular ROS levels in splenic T-cells treated with ANE, which was also attenuated by the presence of NAC. Concordantly, the cellular level of glutathione was diminished by ANE in splenic T-cells pretreated with NAC. Collectively, these results demonstrated that ANE markedly suppressed T-cell activation and Th1 cytokine production, which was mediated, at least in part, by the induction of oxidative stress.