Enhanced oxidative stress and increased mitochondrial mass during Efavirenz-induced apoptosis in human hepatic cells

BACKGROUND AND PURPOSE

Efavirenz (EFV) is widely used in the treatment of HIV-1 infection. Though highly efficient, there is growing concern about EFV-related side effects, the molecular basis of which remains elusive.

EXPERIMENTAL APPROACH

In vitro studies were performed to address the effect of clinically relevant concentrations of EFV (10, 25 and 50 µM) on human hepatic cells.

KEY RESULTS

Cellular proliferation and viability were reduced in a concentration-dependent manner. Analyses of the cell cycle and several cell death parameters (chromatin condensation, phosphatidylserine exteriorization, mitochondrial proapoptotic protein translocation and caspase activation) revealed that EFV triggered apoptosis via the intrinsic pathway. In addition, EFV directly affected mitochondrial function in a reversible manner, inducing a decrease in mitochondrial membrane potential and an increase in mitochondrial superoxide production, followed by a reduction in cellular glutathione content. The rapidity of these actions rules out any involvement of mitochondrial DNA replication, which, until now, was thought to be the main mechanism of mitochondrial toxicity of antiretroviral drugs. Importantly, we also observed an increase in mitochondrial mass, manifested as an elevated cardiolipin content and enhanced expression of mitochondrial proteins, which was not paralleled by an increase in the mtDNA/nuclear DNA copy number ratio. The toxic effect of EFV was partially reversed by antioxidant pretreatment, which suggests ROS generation is involved in this effect.

CONCLUSION AND IMPLICATIONS

Clinically relevant concentrations of EFV were shown to be mitotoxic in human hepatic cells in vitro, which may be pertinent to the understanding of the hepatotoxicity associated with this drug.     

Taurine antagonized oxidative stress injury induced by homocysteine in rat vascular smooth muscle cells

INTRODUCTION Homocysteine is athiol-containing amino acid thatis formed when methionine was converted to cysteine.Plasma homocysteine concentration may increase invarious pathophysiological conditions, including defi-ciency of vitamins such as folic acid, cyanocobalamin,and pyridoxal phosphate etc[1]. Incubation of humanmegakaryocytic cell line DAMI with homocysteine in-hibited respiratory rate and activities of cytochrome coxidase III/ATPase 6, 8 and other respiratory chain en-zymes i…     

Modulation of oxidative/nitrosative stress and mitochondrial protective effect of Semecarpus anacardium in diabetic rats

Objectives Oxidative and nitrosative stress play an important role in the complications of diabetes mellitus. Free radicals are produced when there is an electron leak in the mito‐chondria and a change in the mitochondrial membrane potential. The present study was undertaken to investigate the role of Semecarpus anacardium in protecting the mito‐chondria by modulating the production of reactive oxygen species and reactive nitrogen species in diabetic rats. Methods Diabetes was induced using streptozotocin at a dose of 50 mg/kg body weight and, starting 3 days after the induction, Semecarpus anacardium nut milk extract was administered for 21 days. The same duration of study was used for control, diabetes‐induced and drug control groups, together with a group treated with metformin. After the experimental period, the animals were sacrificed and the levels of superoxide, hydrogen peroxide, nitrate and nitrite were estimated. Changes in mitochondrial membrane potential, intracellular reactive oxygen species and intracellular calcium were also determined. Confocal laser microscopic images were taken for mitochondria isolated from the liver and kidneys. Key findings The results of the study revealed that Semecarpus anacardium was able to decrease the production of reactive oxygen and nitrogen species, and reverse the changes in mitochondrial membrane potential and the influx of calcium into the mitochondria. Conclusions The mitochondrial protective effect may be mediated by scavenging of free radicals and complexing of metal ions by virtue of the antioxidative effect of Semecarpus anacardium.     

SirT3调节氧化应激作用

依赖于烟酰胺腺嘌呤二核苷酸的去乙酰化酶Sirtuin-3(SirT3)是在进化上高度保守的Sirtuin家族成员之一,能对线粒体内相关的乙酰化蛋白脱乙酰基,通过增加活性氧自由基(ROS)清除酶活性和稳定线粒体功能来抑制线粒体内ROS的蓄积。该文综述了SirT3调节因氧化应激诱发的功能性及器质性损伤的作用和机制,为SirT3在相关病理及生理机制的研究提供有益的参考。     

Digitoflavone provokes mitochondrial biogenesis in PC12 cells: A protective approach to oxidative stress

Abstract

Context: Reactive oxygen species (ROS) are known to be one of the main causes of neurodegenerative disorders, and flavonoids play characteristic roles in a variety of biological activities, and specially are known to be antioxidant reagents.

Objective: In this study, we investigated neuroprotective effects of digitoflavone to suppress H₂O₂ -induced cell death in neuron-like PC12 cells.

Material and methods: PC12 cells were pre-treated with digitoflavone for 2?h and then cells were exposed to H₂O₂ for 18?h. The cells’ viability was evaluated by MTT assay. Rhodamine 123 staining was used for the determination of mitochondrial membrane potential (ΔΨ_m). The intracellular ROS aggregation was determined by using 2′,7′-dichlorofluorescein diacetate. Also, the level of mitochondrial biogenesis factors was measured by western blot. The antioxidant capacity of digitoflavone was also determined by measuring reduced glutathione (GSH) level and catalase (CAT) activity quantification.

Results: Digitoflavone significantly elevated cells’ viability at concentrations of 10 and 20?µM. Also, digitoflavone attenuated intracellular level of ROS, and stabilized ΔΨ_m. Moreover, digitoflavone increased phosphorylation of AMP-activated protein kinase (AMPK) and, consequently, elevated mitochondrial biogenesis factors which were reduced after H₂O₂ exposure. We emphasized on the protective effect of digitoflavone through increasing mitochondrial biogenesis by specifically inhibiting AMPK. Antioxidant ability of digitoflavone was indicated by the elevation of GSH level and CAT activity.

Conclusion: As a result, digitoflavone stabilize ΔΨ_m, enhanced cell viability through inducing mitochondrial biogenesis pathway, and increased antioxidant capacity of the cells which lead to better combating the oxidative stress.     

胡黄连甙Ⅱ和神经生长因子对H2O2损伤的PC12细胞的协同保护作用

目的:考察胡黄连甙II和NGF对双氧水损伤细胞的协同保护作用.方法:以CDCFH方法评价活性氧水平,MTT方法、细胞形态观察以及LDH漏出实验评价药物对细胞损伤的修复作用.结果:25 μg/mL胡黄连甙II和2 ng/mL NGF显著改善H2O2诱导的PC12细胞损伤,同时有效降低细胞内ROS.细胞形态改善、细胞活力提高、ROS清除作用均表明胡黄连甙II和NGF协同保护氧化应激对神经细胞的损伤.结论:胡黄连甙II和NGF的协同作用能有效治疗神经性疾病.     

胡黄连甙II和神经生长因子对H_2O_2损伤的PC12细胞的协同保护作用(英文)

目的:考察胡黄连甙II和NGF对双氧水损伤细胞的协同保护作用。方法:以CDCFH方法评价活性氧水平,MTT方法、细胞形态观察以及LDH漏出实验评价药物对细胞损伤的修复作用。结果:25μg/mL胡黄连甙II和2 ng/mL NGF显著改善H2O2诱导的PC12细胞损伤,同时有效降低细胞内ROS。细胞形态改善、细胞活力提高、ROS清除作用均表明胡黄连甙II和NGF协同保护氧化应激对神经细胞的损伤。结论:胡黄连甙II和NGF的协同作用能有效治疗神经性疾病。     

线粒体功能障碍在心血管疾病中作用机制的研究进展

摘要：线粒体是细胞的能量代谢中心,其通过氧化磷酸化产生能量以满足心脏的高能量需求。心脏许多重要的 生理活动,如心肌收缩和细胞内稳态的维持均需要线粒体产生的三磷酸腺苷（ATP）。因此,完整的线粒体结构和功 能是心脏进行正常生理活动的前提和基础。以呼吸链功能异常和ATP合成障碍为特征的线粒体功能障碍是许多心 血管疾病的发病原因。因此,在疾病早期进行线粒体质量控制、减少线粒体功能障碍和氧化应激成为心血管疾病治 疗的新靶标。本文对线粒体功能障碍在心血管疾病中的作用机制以及相关的新型治疗策略展开综述。     

Raloxifene protects endothelial cell function against oxidative stress

Background and purpose:

Maintaining a delicate balance between the generation of nitric oxide (NO) and removal of reactive oxygen species (ROS) within the vascular wall is crucial to the physiological regulation of vascular tone. Increased production of ROS reduces the effect and/or bioavailability of NO, leading to an impaired endothelial function. This study tested the hypothesis that raloxifene, a selective oestrogen receptor modulator, can prevent endothelial dysfunction under oxidative stress.

Experimental approach:

Changes in isometric tension were measured in rat aortic rings. The content of cyclic GMP in aortic tissue was determined by radioimmunoassay. Phosphorylation of endothelial NOS (eNOS) and Akt was assayed by Western blot analysis.

Key results:

In rings with endothelium, ACh-induced relaxations were attenuated by a ROS-generating reaction (hypoxanthine plus xanthine oxidase, HXXO). The impaired relaxations were ameliorated by acute treatment with raloxifene. HXXO suppressed the ACh-stimulated increase in cyclic GMP levels; this effect was antagonized by raloxifene. The improved endothelial function by raloxifene was abolished by ICI 182,780, and by wortmannin or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Raloxifene also protected endothelial cell function against H₂O₂. Raloxifene increased the phosphorylation of eNOS at Ser-1177 and Akt at Ser-473; this effect was blocked by ICI 182,780. Finally, raloxifene was not directly involved in scavenging ROS, and neither inhibited the activity of xanthine oxidase nor stimulated that of superoxide dismutase.

Conclusion and implications:

Raloxifene is effective against oxidative stress-induced endothelial dysfunction in vitro through an ICI 182,780-sensitive mechanism that involves the increased phosphorylation and activity of Akt and eNOS in rat aortae.     

Development of an albuminous reactive oxygen species assay for photosafety evaluation under experimental biomimetic conditions

The generation of reactive oxygen species (ROS) from an ultraviolet (UV)‐exposed chemical can be an experimental indicator of phototoxic potential. The aim of the present study was to develop a new ROS assay using serum albumin to provide photosafety assessment under experimental biomimetic conditions. To assess assay robustness, a validation study on an albuminous ROS (aROS) assay was conducted with a focus on intra‐ and inter‐day precisions and Z’‐factor reflecting both the assay signal‐to‐noise ratio and variation associated with signal measurements. In the aROS assay on quinine HCl (200 μM), a typical phototoxic drug, both intra‐ and inter‐day precisions (coefficient of variation; CV) were found to be below 4%, and the Z’‐factors for singlet oxygen and superoxide suggested a large separation band between samples and blank signals. To evaluate the prediction capacity, the aROS and ROS assays were applied to 21 phototoxins and 10 non‐phototoxic chemicals. Upon aROS assay on these model chemicals, the individual specificity was 100%, and the positive and negative predictivities were found to be 100% and 81.8%, respectively. The aROS assay can be employed for poorly soluble chemicals for which the ROS assay is unavailable. Comparing the ROS assay data, there seemed to be a photochemical transition of some chemicals in albuminous solution. A molecular interaction between albumin and chemical was also assessed by UV and fluorescent spectroscopic analyses, and the results suggested the limited relationship between the albumin–chemical interaction and the photochemical change. The aROS assay may allow photosafety assessment of new drug entities with a wide range of applicability partly under experimental biomimetic conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

氧化应激在糖尿病视网膜病发病机制中的研究进展

糖尿病视网膜病是糖尿病最常见和严重的微血管并发症之一。近年来大量研究表明,氧化应激和糖尿病视网膜病变的发生发展有一定的相关性。高血糖引起体内氧化应激反应,氧化应激又通过损害内皮细胞、线粒体,生成异常代谢通路,促进炎性反应等加重对视网膜组织的损害,造成视网膜缺血、甚至脱离等严重后果;而抗氧化治疗在临床上也取得了一定的成效。本文主要围绕氧化应激和糖尿病视网膜病的发病关系展开综述。     

Retigeric acid B-induced mitophagy by oxidative stress attenuates cell death against prostate cancer cells in vitro

Aim:

Retigeric acid B (RAB), a pentacyclic triterpenic acid from Lobaria kurokawae Yoshim, has been found to induce apoptosis in prostate cancer cells. The aim of this study was to investigate the roles of mitochondrial damage-caused mitophagy in RAB-induced prostate cancer cell death in vitro.

Methods:

Human prostate cancer PC3 and LNCaP cells were tested. Cell viability was analyzed with MTT assay. Cell apoptosis, ROS level and mitochondrial transmembrane potential (mtΔψ) were measured with flow cytometry. Autophagy- and apoptosis-related proteins were studied using Western blotting. GFP-LC3B puncta, mitochondrial swelling and mitophagy were examined morphologically. Quantitative RT-PCR was used to measure LC3B mRNA level, and siRNA was used to knock down LC3BII.

Results:

In both PC3 and LNCaP cells, RAB (15 μmol/L) increased ROS accumulation and decreased mtΔψ in a time-dependent manner. Furthermore, RAB induced mitochondrial swelling and mitophagy, significantly increased LC3B expression and conversion of LC3BI to LC3BII, and the elimination of mitochondria by LC3BII-containing autophagolysosomes. In addition, RAB suppressed the PI3K/Akt/mTOR pathway activation. Pretreatment of PC3 cells with autophagy inhibitor 3-MA (5 mmol/L) or the lysosomal protease inhibitor CQ (10 μmol/L) significantly increased RAB-induced apoptosis. Similar results were obtained in RAB-treated PC3 cells with LC3B knocked down.

Conclusion:

RAB induces mitochondrial damage and mitophagy that attenuates RAB-induced prostate cancer cell death. Thus, suppression of mitophagy might be a potential strategy for improving the chemotherapeutic effects of RAB.     

Inhibitory mechanism against oxidative stress of caffeic acid

The purpose of this article is to summarize the reported antioxidant activities of a naturally abundant bioactive phenolic acid, caffeic acid (CA, 3,4-dihydroxycinnamic acid), so that new avenues for future research involving CA can be explored. CA is abundantly found in coffee, fruits, vegetables, oils, and tea. CA is among the most potential and abundantly found in nature, hydroxycinnamic acids with the potential of antioxidant behavior. Reactive oxygen species produced as a result of endogenous processes can lead to pathophysiological disturbances in the human body. Foods containing phenolic substances are a potential source for free radical scavenging; these chemicals are known as antioxidants. This review is focused on CA’s structure, availability, and potential as an antioxidant along with its mode of action. A brief overview of the literature published about the prooxidant potential of caffeic acid as well as the future perspectives of caffeic acid research is described. CA can be effectively employed as a natural antioxidant in various food products such as oils.     

Opportunities for future therapeutic interventions for hyperoxaluria: targeting oxidative stress

Introduction: Oxalate is a toxic byproduct of metabolism and is normally produced in quantities easily removed from the body. However, under specific circumstances oxalate production is increased resulting in deposition of calcium oxalate (CaOx) crystals in the kidneys as well as other organs causing inflammation and injury. Excessive buildup of crystal deposits in the kidneys causes eventual loss of renal function requiring renal transplantation.

Areas covered: Cellular exposure to CaOx crystals induces the production of reactive oxygen species (ROS) with the involvement of renin-angiotensin aldosterone system (RAAS), mitochondria, and NADPH oxidase. Inflammasomes are activated and pro-inflammatory cytokines, such as IL-1β and IL-18 are produced. We reviewed results of experimental and clinical studies of crystal renal epithelial cell interactions with emphasis on cellular injury and ROS production.

Expert opinion: Treatment should depend upon the level of hyperoxaluria and whether it is associated with CaOx crystal deposition. Persistent low grade or intermittent hyperoxaluria can be treated with antioxidants, free radical scavengers. Hyperoxaluria associated with CaOx crystal deposition will require administration of angiotensin II receptor blockers, and NADPH oxidase or NLRP3 inflammasome inhibitors. DASH-style diet will be beneficial in both cases.     

ROS generated by CYP450, especially CYP2E1, mediate mitochondrial dysfunction induced by tetrandrine in rat hepatocytes

Aim:

Tetrandrine, an alkaloid with a remarkable pharmacological profile, induces oxidative stress and mitochondrial dysfunction in hepatocytes; however, mitochondria are not the direct target of tetrandrine, which prompts us to elucidate the role of oxidative stress in tetrandrine-induced mitochondrial dysfunction and the sources of oxidative stress.

Methods:

Rat primary hepatocytes were isolated by two-step collagenase perfusion. Mitochondrial function was evaluated by analyzing ATP content, mitochondrial membrane potential (MMP) and the mitochondrial permeability transition. The oxidative stress was evaluated by examining changes in the levels of reactive oxygen species (ROS) and glutathione (GSH).

Results:

ROS scavengers largely attenuated the cytotoxicity induced by tetrandrine in rat hepatocytes, indicating the important role of ROS in the hepatotoxicity of tetrandrine. Of the multiple ROS inhibitors that were tested, only inhibitors of CYP450 (SKF-525A and others) reduced the ROS levels and ameliorated the depletion of GSH. Mitochondrial function assays showed that the mitochondrial permeability transition (MPT) induced by tetrandrine was inhibited by SKF-525A and vitamin C (VC), both of which also rescued the depletion of ATP levels and the mitochondrial membrane potential. Upon inhibiting specific CYP450 isoforms, we observed that the inhibitors of CYP2D, CYP2C, and CYP2E1 attenuated the ATP depletion that occurred following tetrandrine exposure, whereas the inhibitors of CYP2D and CYP2E1 reduced the ROS induced by tetrandrine. Overexpression of CYP2E1 enhanced the tetrandrine-induced cytotoxicity.

Conclusion:

We demonstrated that CYP450 plays an important role in the mitochondrial dysfunction induced by the administration of tetrandrine. ROS generated by CYP450, especially CYP2E1, may contribute to the mitochondrial dysfunction induced by tetrandrine.     

氧化应激致心室重构的研究进展

心室重构意味着血管壁中氧化应激水平的提高,当机体受到有害刺激时,氧化和抗氧化之间失衡,导致大量活性氧簇（ ROS）的产生,加速心室重构的进展。近年来,新型抗氧化剂成为治疗或预防心室重构的靶点,观察抗氧化剂治疗在心室重构患者的长期应用是当下趋势。该文就氧化应激与心室重构之间的关系及相关药物治疗进行综述。     

Influence of NADPH oxidase inhibition on oxidative stress parameters in rat hearts

BackgroundThe aim of this study was to assess whether apocynin, an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocker, influences lipid peroxidation TBARS, hydrogen peroxide (H₂O₂) content, protein level, heart edema, tumor necrosis factor a (TNF-α) concentration or the glutathione redox system in heart homogenates obtained from endothelin 1 (ET-1)-induced oxidative stress rats.MethodsExperiments were carried out on adult male Wistar-Kyoto rats. The animals were divided into 4 groups: Group I: saline-treated control; Group II: saline followed by ET-1 (3 μg/kg b.w., iv); Group III: apocynin (5 mg/kg b.w., iv) administered half an hour before saline; Group IV: apocynin (5 mg/kg b.w., iv) administered half an hour before ET-1 (3 μg/kg b.w., iv).ResultsInjection of ET-1 alone showed a significant (p < 0.001) increase in thiobarbituric acid reactive substances (TBARS) and the hydrogen peroxide level (p < 0.01) vs. control, as well as a decrease (p < 0.001) in the GSH level. Apocynin significantly decreased TBARS (p < 0.001) and H₂O₂ (p < 0.05) level (vs. control) as well as improved protein level (p < 0.001) in the heart. Apocynin also prevented ET-1-induced heart edema (p < 0.05). The presence of ET-1 increased the concentration of TNF-α (p < 0.05) while apocynin decreased it (p < 0.05). Our results indicate that ET-1 may induce oxidative stress in heart tissue by reducing the GSH/GSSG ratio, stimulating lipid peroxidation and increasing TNF-α concentration. Apocynin diminished these measures of oxidative stress and TNF-α.ConclusionET-1-induced formation of ROS in the heart is at least partially regulated via NADPH oxidase.     

The effects of methylmercury on mitochondrial function and reactive oxygen species formation in rat striatal synaptosomes are age-dependent.

Methylmercury (MeHg) is especially toxic to the developing central nervous system. In order to understand the reasons for this age-dependent vulnerability, we compared the effects of MeHg on formation of reactive oxygen species (ROS) and mitochondrial function in striatal synaptosomes obtained from rats of various ages. Basal ROS levels were greater, and basal mitochondrial function was lower, in synaptosomes from younger animals, compared to adult animals. MeHg induced ROS formation in synaptosomes from rats of all ages, although the increases were greatest in synaptosomes from the younger animals. MeHg also reduced mitochondrial metabolic function, as assessed by MTT reduction, as well as mitochondrial membrane potential; again, the greatest changes were seen in synaptosomes from early postnatal animals. These age-dependent differences in susceptibility to MeHg are most likely due to a less efficient ROS detoxifying system and lower activity of mitochondrial enzymes in tissue from young animals.     

Dose-dependent protective effect of propofol against mitochondrial dysfunction in ischaemic/reperfused rat heart: role of cardiolipin

BACKGROUND AND PURPOSE:Ischaemia damages to the cardiac mitochondria by increasing generation of reactive oxygen species (ROS) and peroxidation of cardiolipin. The inhibited mitochondrial function leads to the cardiac injury during reperfusion. Propofol (2, 6-diisopropylphenol), an intravenous anaesthetic, has been shown to decrease cardiac ischaemia and reperfusion injury. In the present study, we propose that propofol protects mitochondrial function and decreases cardiac injury by prevention of cardiolipin peroxidation during ischaemia and reperfusion. EXPERIMENTAL APPROACH:After isolation of mitochondria from isolated rat heart perfused on a Langendorff model, various mitochondrial bioenergetic parameters were evaluated such as rates of mitochondrial oxygen consumption, H(2)O(2) production, complex I and III activity as well as the degree of lipid peroxidation and cardiolipin content. The action of propofol was also explored in isolated mitochondria. And the effect of cardiolipin was evaluated by fusing cardiolipin liposome with mitochondria. KEY RESULTS:Propofol treatment had strong dose-dependent protection attenuating these parameters alterations in reperfused rat heart and isolated mitochondria. Additionally, cardiolipin treatment had the same protective effect, compared with propofol treatment at high concentration. CONCLUSIONS AND IMPLICATIONS:The protective effect of propofol appears to be due, at least in part, as a chemical uncoupler, to the interruption of the vicious circle of ROS-cardiolipin-complexes of the respiratory chain-ROS through preserving the content and integrity of cardiolipin molecules by ROS attack. These findings may provide an explanation for some of the factors responsible for cardioprotection and one approach exploring an available antioxidant.     

Isoniazid-induced apoptosis in HepG2 cells: Generation of oxidative stress and Bcl-2 down-regulation

Isoniazid (INH) is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. However it has a serious limitation of being hepatotoxic. Delineating the mechanism underlying INH-induced hepatotoxicity may be beneficial in devising ways to counteract its toxic manifestations. Studies in human hepatoma HepG2 cells have indicated that INH exposure causes induction of apoptosis. This study was aimed at identifying the key components/pathways of the INH-induced apoptotic pathway using HepG2 cells. HepG2 cells were exposed to increasing concentrations of INH (6.5, 13, 26, and 52?mM). Hydrogen peroxide (0.3?mM) served as positive control. After incubating for specific time intervals cells were harvested and evidences of cytotoxicity, oxidative stress, and apoptosis were sought. The findings indicated that INH exposure causes increased ROS generation along with alteration in levels of enzymatic antioxidants such as Superoxide dismutase, Catalase, and Glucose-6-Phosphate dehydrogenase. Altered Bcl-2/Bax content, cytochrome-c translocation, caspase activation, and DNA fragmentation emphasized involvement of apoptosis.