Antioxidant Agent N-acetylcysteine Research Articles

Repurposing the antioxidant and anti-inflammatory agent N-acetyl cysteine for treating COVID-19.

Although several considerations have been raised suggesting a beneficial effect of N-acetyl cysteine (NAC) for the treatment of severe acute respiratory syndrome coronavirus 2 infection, there is currently no clinical evidence that NAC truly prevents coronavirus disease 2019 (COVID-19), reduces the severity of the disease, or improves the outcome. Appropriately designed clinical trials are warranted to prove or disprove a therapeutic effect of NAC for COVID-19 patients.

N-Acetylcysteine Reduces Skeletal Muscles Oxidative Stress and Improves Grip Strength in Dysferlin-Deficient Bla/J Mice.

Dysferlinopathy is an autosomal recessive muscular dystrophy resulting from mutations in the dysferlin gene. Absence of dysferlin in the sarcolemma and progressive muscle wasting are hallmarks of this disease. Signs of oxidative stress have been observed in skeletal muscles of dysferlinopathy patients, as well as in dysferlin-deficient mice. However, the contribution of the redox imbalance to this pathology and the efficacy of antioxidant therapy remain unclear. Here, we evaluated the effect of 10 weeks diet supplementation with the antioxidant agent N-acetylcysteine (NAC, 1%) on measurements of oxidative damage, antioxidant enzymes, grip strength and body mass in 6 months-old dysferlin-deficient Bla/J mice and wild-type (WT) C57 BL/6 mice. We found that quadriceps and gastrocnemius muscles of Bla/J mice exhibit high levels of lipid peroxidation, protein carbonyls and superoxide dismutase and catalase activities, which were significantly reduced by NAC supplementation. By using the Kondziela’s inverted screen test, we further demonstrated that NAC improved grip strength in dysferlin deficient animals, as compared with non-treated Bla/J mice, without affecting body mass. Together, these results indicate that this antioxidant agent improves skeletal muscle oxidative balance, as well as muscle strength and/or resistance to fatigue in dysferlin-deficient animals.

GSTZ1 deficiency promotes hepatocellular carcinoma proliferation via activation of the KEAP1/NRF2 pathway

BackgroundGlutathione S-transferase zeta 1 (GSTZ1) is the penultimate enzyme in phenylalanine/tyrosine catabolism. GSTZ1 is dysregulated in cancers; however, its role in tumorigenesis and progression of hepatocellular carcinoma (HCC) is largely unknown. We aimed to assess the role of GSTZ1 in HCC and to reveal the underlying mechanisms, which may contribute to finding a potential therapeutic strategy against HCC.MethodsWe first analyzed GSTZ1 expression levels in paired human HCC and adjacent normal tissue specimens and the prognostic effect of GSTZ1 on HCC patients. Thereafter, we evaluated the role of GSTZ1 in aerobic glycolysis in HCC cells on the basis of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Furthermore, we assessed the effect of GSTZ1 on HCC proliferation, glutathione (GSH) concentration, levels of reactive oxygen species (ROS), and nuclear factor erythroid 2-related factor 2 (NRF2) signaling via gain- and loss- of GSTZ1 function in vitro. Moreover, we investigated the effect of GSTZ1 on diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) induced hepatocarcinogenesis in a mouse model of HCC.ResultsGSTZ1 was downregulated in HCC, thus indicating a poor prognosis. GSTZ1 deficiency significantly promoted hepatoma cell proliferation and aerobic glycolysis in HCC cells. Moreover, loss of GSTZ1 function depleted GSH, increased ROS levels, and enhanced lipid peroxidation, thus activating the NRF2-mediated antioxidant pathway. Furthermore, Gstz1 knockout in mice promoted DEN/CCl4-induced hepatocarcinogenesis via activation of the NRF2 signaling pathway. Furthermore, the antioxidant agent N-acetylcysteine and NRF2 inhibitor brusatol effectively suppressed the growth of Gstz1-knockout HepG2 cells and HCC progression in Gstz1−/− mice.ConclusionsGSTZ1 serves as a tumor suppressor in HCC. GSH depletion caused by GSTZ1 deficiency elevates oxidative stress, thus constitutively activating the NRF2 antioxidant response pathway and accelerating HCC progression. Targeting the NRF2 signaling pathway may be a promising therapeutic approach for this subset of HCC.

Decitabine shows synergistic effects with arsenic trioxide against myelodysplastic syndrome cells via endoplasmic reticulum stress-related apoptosis

Most of the International Prognostic Scoring System (IPSS) high-risk patients with myelodysplastic syndrome partly responded to hypomethylating therapy even with transient remission, while arsenic trioxide (ATO) had partial effect in...

Endoplasmic reticulum stress-dependent oxidative stress mediated vascular injury induced by silica nanoparticles in vivo and in vitro

The safety concern on silica nanoparticles (SiNPs) is rapidly expanding with its increased production for industrial, environmental, and pharmaceutical applications. We have reported SiNPs induced endothelial dysfunction, which potentially precedes atherosclerosis and ultimately contributes to cardiovascular disease. However, its molecular mechanisms haven't been clearly determined. Here, we observed the subchronic exposure of SiNPs induced vascular injury, accompanied by oxidative stress, ER stress and apoptosis in the artery. Further, their detailed interactions in SiNPs-induced vascular injury were investigated in vitro. SiNPs were internalized and deposited the intracellular ER region of human umbilical vein endothelial cells (HUVECs), and triggered the activation of ER stress as validated by the up-regulated PERK/eIF2α/ATF4, IRE1/XBP1 and ATF6 pathways. Mechanistic analyses by using ER stress inhibitor (4-PBA or Salubrinal) and antioxidant agent N-acetylcysteine (NAC) confirmed an ER stress-dependent oxidative stress and apoptosis induced by SiNPs, which mediated the consequent vascular endothelial injury, ultimately contributing to vascular disease.

Emodin Promotes Apoptosis of Human Endometrial Cancer Through Regulating the MAPK and PI3K/ AKT Pathways.

Emodin, a major component of rhubarb, has anti-tumor effects in a variety of cancers, influencing multiple steps of tumor development through modulating several signaling pathways. The aim of this study is to examine the effect of emodin on cell apoptosis and explore the underlying mechanisms in human endometrial cancer cells. Here we report that emodin can inhibit KLE cell proliferation and induce apoptosis in a time- and dose-dependent manner. Western blot assay found that emodin was involved in MAPK and PI3K/Akt signaling pathways. Specifically, emodin significantly suppressed the phosphorylation of AKT, and enhanced the phosphorylation of MAPK pathways. Furthermore, the generation of reactive oxygen species (ROS) was up-regulated in KLE cells upon treatment with emodin, while the anti-oxidant agent N-acetyl cysteine (NAC) can inhibit emodin-induced apoptosis and promote the activation of AKT and Bcl-2. Taken together, we revealed that emodin may induce apoptosis in KLE cells through regulating the PI3K/AKT and MAPK signaling pathways, indicating the importance of emodin as an anti-tumor agent.

Effect of rutin on cisplatin-induced damage in human mesangial cells via apoptotic pathway.

Cisplatin (CP) is one of the most effective and widely used compounds in the treatment of disease, including cancer, but is known to induce toxicity in patients. Rutin (RUT) is a flavonoid glycoside from Sophora japonica L. that has been shown to possess antioxidative, anti-inflammatory, and antiviral properties. RUT is also known to attenuate cardiotoxicity, isoproterenol-induced cardiac fibrosis, and ischemia/reperfusion-associated hemodynamic alteration, and prevents high glucose-induced renal glomerular endothelial hyperpermeability. In this study, we investigated the effect of RUT on CP-induced nephrotoxicity. CP was used to induce toxicity in human mesangial cells (HMCs), HMCs were pretreated with different concentrations of RUT before being exposed to 10 μg/mL of CP. A positive group was pretreated with antioxidant agent N-acetylcysteine prior to CP administration. At doses between 12.5 and 25 μM, RUT prevented CP-induced reduction in cell viability. Treatment with RUT suppressed intracellular reactive oxygen species and malonic dialdehyde levels and inhibited cell apoptosis. RUT reversed the CP-induced upregulation of p53, cleaved-caspase-3, and increased pro-caspase-3 and pro-caspase-9 levels. In conclusion, the RUT can relieve CP-induced nephrotoxicity by inhibiting the p53/caspase signaling pathway.

A critical role for very long-chain fatty acid elongases in oleic acid-mediated Saccharomyces cerevisiae cytotoxicity

Elongases FEN1/ELO2 and SUR4/ELO3 are important enzymes involved in the elongation of long-chain fatty acids (LCFAs) to very long-chain fatty acids (VLCFAs) in Saccharomyces cerevisiae. The molecular mechanism of the involvement of these elongases in lipotoxicity is unclear. In the present study, we investigated the role of VLCFA elongases in oleic acid-mediated yeast cytotoxicity. The spot test showed that yeast strains with the deletion of ELO2 or ELO3 were strikingly sensitive to oleic acid, while there was no change on the growth of strain with deleted ELO1 which was involved in the elongation of C14 fatty acid (FA) to C16 FA. By using GC–MS, the unsaturation index was increased in elo2△ and elo3△ mutants after treatment with oleic acid (OLA). However, the proportion of VLCFAs was increased in response to OLA in the wild-type strain. The growth inhibition of elo2△ and elo3△ could be partially rescued by two commonly used antioxidant agents N-acetyl cysteine (NAC) and Ascorbic acid (VC). The further study showed that exposure to excess OLA led to an increase in the levels of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS), and a decline in the quantity of reduced glutathione (GSH) in both the wild type and mutant strains. However, the antioxidant enzyme activities of superoxide dismutase (SOD) and catalase (CAT) were increased in the wild type and elo1△ strains, while they were significantly decreased in the mutants of elo2△ and elo3△ after treated with excess OLA. Thus, oxidative damage mainly contributed to the cell death induced by OLA in ole2△ and ole3△. Taken together, although disruption of ELO2 or ELO3 did not affect the cellular lipid unsaturation, they altered the distribution and propotion of cellular VLCFAs, leading to the cell membrane impairment, which augmented the ability of OLA to permeabilize the plasma membrane. The data suggest that the very long-chain fatty acids elongases ELO2 and ELO3 play important roles in lipotoxic cell death induced by OLA through maintaining a balanced FA composition in plasma membrane.

The Neuroprotective Effect of Conditioned Medium from Human Adipose-Derived Mesenchymal Stem Cells is Impaired by N-acetyl Cysteine Supplementation.

Oxidative stress is a common feature in neurodegenerative diseases associated with neuroinflammation, and therefore, has been proposed as a key target for novel therapies for these diseases. Recently, adipose-derived stem cell (ASC)-based cell therapy has emerged as a novel strategy for neuroprotection. In this study, we evaluate the therapeutic role of ASC-conditioned medium (ASC-CM) against H2O2-induced neurotoxicity in a new in vitro model of ec23/brain-derived neurotrophic factor (BDNF)-differentiated human SH-SY5Y neuron-like cells (SH-SY5Yd). In the presence of ASC-CM, stressed SH-SY5Yd cells recover normal axonal morphology (with an almost complete absence of H2O2-induced axonal beading), electrophysiological features, and cell viability. This beneficial effect of ASC-CM was associated with its antioxidant capacity and the presence of growth factors, namely, BDNF, glial cell line-derived neurotrophic factor, and transforming growth factor β1. Moreover, the neuroprotective effect of ASC-CM was very similar to that obtained from treatment with BDNF, an essential factor for SH-SY5Yd cell survival. Importantly, we also found that the addition of the antioxidant agent N-acetyl cysteine to ASC-CM abolished its restorative effect; this was associated with a strong reduction in reactive oxygen species (ROS), in contrast to the moderate decrease in ROS produced by ASC-CM alone. These results suggest that neuronal restorative effect of ASC-CM is associated with not only the release of essential neurotrophic factors, but also the maintenance of an appropriate redox state to preserve neuronal function.

Apigenin oxidovanadium(IV) cation interactions. Synthesis, spectral, bovine serum albumin binding, antioxidant and anticancer studies

Abstract Continuing and expanding our previous work on flavonoid oxidovanadium(IV) (VO) metal complexes as possible anti-cancer agents, the VOapigenin compound was synthesized and characterized. An “acetylacetone-like” coordination through the C O and O moieties of the ligand to the metal center with one apigenin ligand per metal ion was assumed using different spectroscopies and elemental analysis as well as thermal measurements. The vibrational experimental spectrum of VOapigenin was supported by theoretical calculations. According to the structure of the flavonoid it exerted mild antioxidant properties that were enhanced by metal coordination. The compounds showed moderate anticancer activity on lung A549 and cervix HeLa cancer cell lines, displaying an incubation time dependent behavior. Cellular increase of reactive oxygen species (ROS) and glutathione depletion have been measured upon incubation with the compounds. These cell killing activities were reverted when natural antioxidants were incubated with the compounds and the addition of the antioxidant agent N-acetylcysteine generated depletion of the cellular ROS levels. Therefore, a stress oxidative mechanism of action has been assumed. Moreover, the compounds showed no toxicity against Artemia salina and were not mutagenic. Both apigenin and the complex could be transported and stored by bovine serum albumin with similar binding constants and mechanisms than other VOflavonoid complexes.

Pathological concentrations of homocysteine increases IL-1β production in macrophages in a P2X7, NF-ĸB, and erk-dependent manner.

Elevated plasma levels of homocysteine (Hcy) are associated with the development of coronary artery disease (CAD), peripheral vascular disease, and atherosclerosis. Hyperhomocysteinemia is likely related to the enhanced production of pro-inflammatory cytokines including IL-1β. However, the mechanisms underlying the effects of Hcy in immune cells are not completely understood. Recent studies have established a link between macrophage accumulation, cytokine IL-1β, and the advance of vascular diseases. The purpose of the present study is to investigate the effects of Hcy on IL-1β secretion by murine macrophages. Hcy (100 μM) increases IL-1β synthesis via enhancement of P2X7 expression and NF-ĸB and ERK activation in murine macrophages. In addition, the antioxidant agent N-acetylcysteine (NAC) reduces NF-κB activation, ERK phosphorylation, and IL-1β production in Hcy-exposed macrophages, indicating the importance of ROS in this pro-inflammatory process. In summary, our results show that Hcy may be involved in the synthesis and secretion of IL-1β via NF-ĸB, ERK, and P2X7 stimulation in murine macrophages.

The effects of n-acetylcysteine and/or deferoxamine on manic-like behavior and brain oxidative damage in mice submitted to the paradoxal sleep deprivation model of mania

Bipolar disorder (BD) is a severe psychiatric disorder associated with social and functional impairment. Some studies have strongly suggested the involvement of oxidative stress in the pathophysiology of BD. Paradoxal sleep deprivation (PSD) in mice has been considered a good animal model of mania because it induces similar manic-like behavior, as well as producing the neurochemical alterations which have been observed in bipolar patients. Thus, the objective of the present study was to evaluate the effects of the antioxidant agent's n-acetylcysteine (Nac) and/or deferoxamine (DFX) on behavior and the oxidative stress parameters in the brains of mice submitted to the animal model of mania induced by PSD. The mice were treated for a period of seven days with saline solution (SAL), Nac, DFX or Nac plus DFX. The animals were subject to the PSD protocol for 36 h. Locomotor activity was then evaluated using the open-field test, and the oxidative stress parameters were subsequently evaluated in the hippocampus and frontal cortex of mice. The results showed PSD induced hyperactivity in mice, which is considered a manic-like behavior. In addition to this, PSD increased lipid peroxidation and oxidative damage to proteins, as well as causing alterations to antioxidant enzymes in the frontal cortex and hippocampus of mice. The Nac plus DFX adjunctive treatment prevented both the manic-like behavior and oxidative damage induced by PSD. Improving our understanding relating to oxidative damage in biomolecules, and the antioxidant mechanisms presented in the animal models of mania are important in helping to improve our knowledge concerning the pathophysiology and development of new therapeutical treatments for BD.

Positive effects of an oral supplementation by Glisodin, a gliadin-combined SOD-rich melon extract, in an animal model of dietary-induced oxidative stress

We investigated the potential protective effects of two antioxidant molecules: Glisodin, a gliadin combined copper-zinc superoxide dismutase SOD (Cu,Zn SOD)-rich melon extract, SOD is a known enzyme that has been best studied as a regulator of antioxidant defence, and an antioxidant agent N-acetylcysteine (NAC). Glisodin, given orally to rats fed a chow diet, as 180 U/d during 2 weeks in a preconditioning treatment, and then for 8 weeks, combined to a high fat/high fructose diet (HF/HFr), had more positive effects than NAC (100 mg/d), not only on oxidative stress parameters, but also on features of the metabolic-syndrome. DNA oxidative damages, lipid peroxidation, and fasting glycaemia were lower in rats receiving Glisodin than in those supplemented by NAC. In addition, insulin sensitivity was improved and mesenteric fat was significantly lower in rats fed the Fr/Fe diet plus Glisodin than in animals fed NAC supplementation. These data suggest potential beneficial effects of oral Glisodin supplementation in preventing metabolic alterations related to the metabolic syndrome.

N-acetylcysteine chemoprotection without decreased cisplatin antitumor efficacy in pediatric tumor models.

Decreasing oxidative damage with the antioxidant agent N-acetylcysteine (NAC) can block the side effects of chemotherapy, but may diminish anti-tumor efficacy. We tested the potential for interactions of high dose NAC against a minimally effective cisplatin chemotherapy regimen in rat models of human pediatric cancers. Athymic rats received subcutaneous implantation of human SK-N-AS neuroblastoma cells or intra-cerebellar implantation of human D283-MED medulloblastoma cells. Rats were untreated or treated with cisplatin (3 or 4 mg/kg IV) with or without NAC (1,000 mg/kg IV) 30 min before or 4 h after cisplatin treatment. Blood urea nitrogen (BUN) and tumor volumes were measured. Cisplatin decreased the growth of SK-N-AS neuroblastoma subcutaneous tumors from 17.7 ± 4.9 to 6.4 ± 2.5 fold over baseline 2 weeks after treatment (P < 0.001). Pretreatment with NAC decreased cisplatin efficacy, while 4 h delayed NAC did not significantly affect cisplatin anti-tumor effects (relative tumor volume 6.8 ± 2.0 fold baseline, P < 0.001). In D283-MED medulloblastoma brain tumors, cisplatin decreased final tumor volume to 3.9 ± 2.3 mm(3) compared to untreated tumor volume of 45.9 ± 38.7 (P = 0.008). Delayed NAC did not significantly alter cisplatin efficacy (tumor volume 6.8 ± 8.1 mm(3), P = 0.014 versus control). Cisplatin was minimally nephrotoxic in these models. NAC decreased cisplatin-induced elevations in BUN (P < 0.02). NAC chemoprotection did not alter cisplatin therapy, if delayed until 4 h after chemotherapy. These data support a Phase I/II clinical trial of delayed NAC to reduce ototoxicity in children with localized pediatric cancers.

Abstract 839: N-acetylcysteine chemoprotection without decreased cisplatin antitumor efficacy in pediatric tumor models

Abstract PURPOSE. Cisplatin chemotherapy is used in the treatment of many pediatric cancers, but has significant toxicities that impact patient quality of life. Decreasing oxidative damage with the anti-oxidant agent N-acetylcysteine (NAC) can reduce cisplatin side effects; however its use is limited by concerns about decreased anti-tumor efficacy. This study evaluated the effect of the timing of NAC on cisplatin efficacy in vitro and in rat models of human pediatric cancers. METHODS. Female athymic nude rats received subcutaneous implantation of human SK-N-AS neuroblastoma cells or intra-cerebellar implantation of human D283-MED medulloblastoma cells. Rats were untreated or treated with cisplatin (3 or 4 mg/kg IV) with or without NAC (1000 mg/kg IV) 30 min before or 4 h after cisplatin treatment. Blood urea nitrogen (BUN) and tumor volumes were measured. Viability and apoptosis were assessed in vitro in both cell types. RESULTS. NAC was protective against cisplatin toxicity in both SK-N-AS and D283-MED cells in vitro if added within 2 h after chemotherapy. Untreated SK-N-AS neuroblastoma subcutaneous tumor volumes increased by 17.7 ± 4.9 fold over 2 weeks, with final tumor weight of 4.3 ± 1.6 g. Cisplatin decreased relative tumor growth to 6.4 ± 2.5 fold over baseline (P&amp;lt;0.0001) with final tumor weight of 1.7 ± 0.9 g. Pretreatment with NAC significantly decreased cisplatin efficacy (tumor volume 15.0 ± 4.8 fold over baseline, not different from untreated control), while 4 h delayed NAC did not significantly affect cisplatin anti-tumor effects (relative tumor volume 6.8 ± 2.0 fold baseline, P&amp;lt;0.001). For the D283-MED medulloblastoma brain tumors, cisplatin decreased final tumor volume to 3.9 ± 2.3 mm3 compared to untreated tumor volume of 45.9 ± 38.7 (P = 0.008). When NAC was given prior to cisplatin tumor volume did not differ from untreated control (13.3 ± 23.1 mm3, P = 0.060). Delayed NAC was not significantly different than cisplatin alone (tumor volume 6.8 ± 8.1 mm3, P = 0.014 versus control). Two doses of cisplatin increased BUN levels to 57.3 ±19.9 mg/dL at 7 days after treatment, compared to 18.9 ± 2.0 in untreated control rats (P&amp;lt;0.001). Both pretreatment or delayed treatment with NAC decreased BUN compared to cisplatin alone (25.3 ± 6.8 and 27.9 ± 7.5 respectively, P&amp;lt;0.001). We conclude that NAC protects against kidney toxicity without impacting cisplatin therapy, if delayed until 4 h after chemotherapy. These data support a clinical trial of delayed NAC to reduce chemotherapy toxicities in children with cancer. Citation Format: Leslie L. Muldoon, Y. Jeffrey Wu, Michael A. Pagel, Kathleen A. Beeson, Edward A. Neuwelt. N-acetylcysteine chemoprotection without decreased cisplatin antitumor efficacy in pediatric tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 839. doi:10.1158/1538-7445.AM2014-839

Genotoxic Agents Promote the Nuclear Accumulation of Annexin A2: Role of Annexin A2 in Mitigating DNA Damage

Annexin A2 is an abundant cellular protein that is mainly localized in the cytoplasm and plasma membrane, however a small population has been found in the nucleus, suggesting a nuclear function for the protein. Annexin A2 possesses a nuclear export sequence (NES) and inhibition of the NES is sufficient to cause nuclear accumulation. Here we show that annexin A2 accumulates in the nucleus in response to genotoxic agents including gamma-radiation, UV radiation, etoposide and chromium VI and that this event is mediated by the nuclear export sequence of annexin A2. Nuclear accumulation of annexin A2 is blocked by the antioxidant agent N-acetyl cysteine (NAC) and stimulated by hydrogen peroxide (H2O2), suggesting that this is a reactive oxygen species dependent event. In response to genotoxic agents, cells depleted of annexin A2 show enhanced phospho-histone H2AX and p53 levels, increased numbers of p53-binding protein 1 nuclear foci and increased levels of nuclear 8-oxo-2′-deoxyguanine, suggesting that annexin A2 plays a role in protecting DNA from damage. This is the first report showing the nuclear translocation of annexin A2 in response to genotoxic agents and its role in mitigating DNA damage.

The metabolic regulator PGC‐1α links anti‐cancer cytotoxic chemotherapy to reactivation of hepatitis B virus

Patients with chronic hepatitis B virus (HBV) infection are at an increased risk for a severe and a potentially fatal viral reactivation following anti-cancer therapy. The molecular mechanism for this induction of HBV expression is still unclear. Here, we show that treating hepatoma cell line expressing HBV with various anti-cancer cytotoxic agents results in a significant up-regulation of HBV expression. This HBV induction is at the transcriptional level and is time dependent. Interestingly, treating hepatoma cells with anti-cancer cytotoxic agents results in a robust induction of peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), a metabolic and energy regulator that is normally induced in the liver under starvation conditions and that has been previously shown to strongly coactivate HBV transcription. Most importantly, HBV up-regulation following anti-cancer therapy depends on PGC-1α induction, because PGC-1α knock-down abolishes HBV induction. Finally, pretreatment of HBV-expressing cells with the antioxidant agent N-acetylcysteine attenuates the induction of both PGC-1α and HBV in response to anti-cancer treatment, suggesting that chemotherapy-associated PGC-1α induction is mediated by cellular oxidative stress that ultimately leads to HBV up-regulation. We conclude that cytotoxic anti-cancer chemotherapy has a direct and an immune system-independent effect on HBV gene expression, which is mediated by PGC-1α. Our results attribute to this metabolic regulator an unexpected role in linking anti-cancer treatment to HBV reactivation and make PGC-1α a potential target for future anti-HBV therapy, especially under conditions in which it is robustly induced, such as following anti-cancer treatment.

Dopamine oxidation facilitates rotenone-dependent potentiation of N-methyl-d-aspartate currents in rat substantia nigra dopamine neurons

Rotenone is a mitochondrial poison that causes dopamine cell death and is used as a model of Parkinson's disease in rodents. Recently, we showed that rotenone augments currents evoked by N-methyl-D-aspartate (NMDA) by relieving voltage-dependent Mg(2+) block in rat substantia nigra compacta (SNC) dopamine neurons. Because rotenone is well known to generate reactive oxygen species (ROS), we conducted the present experiments to evaluate the role of ROS in mediating the effect of rotenone on NMDA current augmentation. Using patch pipettes to record whole-cell currents from SNC neurons in slices of rat brain, we found that the ability of rotenone (100 nM) to increase NMDA (3-30 μM) current was antagonized by the antioxidant agent n-acetylcysteine (1 mM). In contrast, mercaptosuccinate (1 mM), which blocks glutathione peroxidase and raises tissue levels of H(2)O(2), mimicked rotenone by augmenting inward currents evoked by NMDA. Because oxidation of dopamine can also generate ROS, we explored the role of dopamine on this action of rotenone. We prepared dopamine-depleted midbrain slices from rats that had been pretreated with reserpine (5 mg/kg ip) and alpha-methyl-para-tyrosine (AMPT, 250 mg/kg ip). Dopamine depletion blocked the ability of rotenone (100 nM) to increase inward current evoked by NMDA (30 μM). Rotenone-dependent augmentation of NMDA current was also blocked by the monoamine oxidase inhibitor pargyline (100 μM) in slices prepared from normal rats. In contrast, the dopamine precursor levodopa potentiated the action of rotenone on NMDA current. These results suggest that ROS and/or dopamine oxidation products mediate the ability of rotenone to potentiate NMDA currents. Because excessive NMDA receptor stimulation can produce excitotoxicity, our results suggest that oxidative metabolism of dopamine might facilitate the neurotoxicity of rotenone.

Shikonin Induces Apoptosis through Reactive Oxygen Species/Extracellular Signal-Regulated Kinase Pathway in Osteosarcoma Cells

Shikonin, a major ingredient in the Chinese traditional herb Lithospermum erythrorhixon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we delineated the molecular mechanisms of shikonin in the apoptosis of 143B osteosarcoma cells. Shikonin reduced the cell viability of 143B cells in a dose- and time-dependent manner. The IC(50) at 24 h and 48 h for 143B cells was 4.55 and 2.01microM, respectively. A significantly elicited hypodiploid cell population was found in cells treated with 2, 4, and 8microM shikonin for 24 h. Moreover, treatment with shikonin induced reactive oxygen species (ROS) generation, increased extracellular signal-regulated kinase (ERK) phosphorylation, decreased B-cell lymphoma-2 (Bcl2) expression, and was accompanied by poly(ADP-ribose) polymerase (PARP) cleavage. Pretreatment with the antioxidant agent N-acetyl cysteine (NAC) not only reversed shikonin-induced ROS generation but also significantly attenuated the cytotoxic effects of shikonin in 143B cells. Furthermore, NAC attenuated shikonin-induced ERK phosphorylation. Taken together, our results reveal that shikonin increased ROS generation and ERK activation, and reduced Bcl2, which consequently caused the cells to undergo apoptosis. Therefore, shikonin may be a promising chemotherapeutic agent for osteosarcoma treatment.

261 EVALUATION OF TWO GAS MIXTURES, ADDITION OF N-ACETYLCYSTEINE, AND LIPOPEROXIDATION LEVELS IN PORCINE EMBRYOS PRODUCED IN VITRO

In vitro embryo development can be affected by many different factors, such as incubation atmosphere, which can cause oxidative stress. In this study the effect of 2 gas mixtures and the addition of the antioxidant agent N-acetylcysteine (NAC) on porcine embryos produced in vitro and lipoperoxidation (LPO) were evaluated. Ovaries collected from slaughtered gilts were aspirated to obtain oocytes, to be matured and fertilized in vitro. In experiment 1 (E1), putative zygotes were cultured in NCSU-23 medium at 38.5°C in humidified air and randomly allocated to each of the following atmospheres: conventional atmosphere (CA: 5% CO2 in air) and modified atmosphere (MA: 5% CO2, 5% O2, 90% N2). In experiment 2 (E2), zygotes were incubated in CA or MA and cultured in NCSU-23 with 2.5 mM NAC. In experiment 3 (E3), embryos produced in E1 and E2 were used to evaluate LPO with the TBARS method: embryos were sonicated, then 300 μL of distilled water and 680 μL of thiobarbituric acid were added and heated at 95°C for 20 min in a water bath. The resulting reaction was read by spectrophotometry at 532 nm. Final concentration was expressed in micromoles of TBARS/embryo (μMTE). Five days after IVF, embryos were evaluated to determine their development to the morula stage. Data from each experiment were transformed using arcsine square root function before analysis, and means were compared using ANOVA. For E1, 954 oocytes were used, 475 for CA and 479 for MA, in 5 replicates. The percentage of cleaved embryos was significantly higher in MA (300/479, 63%) compared with CA (245/475, 52%) (P &lt; 0.05). The percentage of morulae obtained was higher in MA (127/479, 27%) than in CA (80/475, 17%) (P &lt; 0.05). In E2, 936 oocytes were used, 470 for CA NAC and 466 for MA NAC, in 5 replicates. There were no differences in the percentages of cleaved embryos in MA NAC (250/466, 54%) compared with CA NAC (243/470, 52%), and morulae in MA NAC (86/466, 18%) compared with CA NAC (81/470, 17%) (P &gt; 0.05). In E3, during the first 24 h of culture the embryos in CA NAC had lower LPO concentrations (0.029μMTE) (P &lt; 0.05) than in CA (0.059μMTE), MA (0.057μMTE), and MA NAC (0.054μMTE). In morulae, a higher concentration of LPO was observed in the CA group (0.103μMTE) compared with MA (0.083μMTE), CA NAC (0.085μMTE), and MA NAC (0.081μMTE). A correlation analysis between morulae development and LPO in CA and MA, with or without NAC, did not show a significant relationship (r = 0.20; P &gt; 0.05). It is concluded that MA improves the development of in vitro-derived porcine embryos; that NAC treatment did not cause differences in any stage of embryo development, probably meaning that the embryo has its own defensive mechanisms against LPO, and therefore that NAC is not a good antioxidant for porcine embryos, or that it does not work well at the dosage and/or in the culture medium used; that the embryos exposed to CA and MA showed higher LPO that the groups with NAC, indicating that the addition of NAC tends to decrease LPO; and that LPO could be a good indicator of embryo viability. We thank CONACYT Mexico for the graduate student’s scholarship.