Optimizing the energy status of skin cells during solar radiation.

Ionizing- and ultraviolet-radiation cause cell damage or death by directly altering DNA and protein structures and by production of reactive oxygen species (ROS) and reactive carbonyl species (RCS). These processes disrupt cellular energy metabolism at multiple levels. The formation of DNA strand breaks activates signaling pathways that consume NAD, which can lead to the depletion of cellular ATP. Poly(ADP)-ribose polymerase (PARP-1) is the enzyme responsible for much of the NAD degradation following DNA damage, although numerous other PARPs have been discovered recently that await functional characterization. Studies on mouse epidermis in vivo and on human cells in culture have shown that UV-B radiation provokes the transient degradation of NAD and the synthesis of ADP-ribose polymers by PARP-1. This enzyme functions as a component of a DNA damage surveillance network in eukaryotic cells to determine the fate of cells following genotoxic stress. Additionally, the activation of PARP-1 results in the activation of a nuclear proteasome that degrades damaged nuclear proteins including histones. Identifying approaches to optimize these responses while maintaining the energy status of cells is likely to be very important in minimizing the deleterious effects of solar radiation on skin.     

Monitoring the Glutathione Redox Reaction in Living Human Cells by Combining Metabolic Labeling with Heteronuclear NMR

The glutathione (GSH) redox reaction is critical for defense against cellular reactive oxygen species (ROS). However, direct and real‐time monitoring of this reaction in living mammalian cells has been hindered by the lack of a facile method. Herein, we describe a new approach that exploits the GSH biosynthetic pathway and heteronuclear NMR. [U‐¹³C]‐labeled cysteine was incorporated into GSH in U87 glioblastoma cells, and the oxidation of GSH to GSSG by a ROS‐producing agent could be monitored in living cells. Further application of the approach to cells resistant to temozolomide (TMZ), an anti‐glioblastoma drug, suggested a possible new resistance mechanism involving neutralization of ROS. This result was corroborated by the observation of up‐regulation of glutathione peroxidase 3 (GPx3). This new approach could be easily applied to redox‐dependent signaling pathways and drug resistance involving ROS.     

多壁碳纳米管诱导A549细胞氧化应激与去极化线粒体膜电位

以人肺上皮细胞系A549为模型细胞, 探讨多壁碳纳米管的细胞毒性效应及其机制. A549细胞暴露于不同浓度(0~300 μg/mL)的多壁碳纳米管后, 用MTT比色法检测细胞活力和Hoechst 33342染色法观察细胞形态; 用活性氧(ROS)敏感探针2',7'-二氯荧光素二乙酸酯(DCFH-DA)结合流式细胞仪检测细胞内ROS水平; 用荧光探针JC-1结合激光共聚焦显微镜检测细胞线粒体膜电位ΔΨm的变化; 用免疫荧光和蛋白印迹法检测细胞氧化应激敏感蛋白血红素氧合酶-1(HO-1)的表达水平. 结果表明, 多壁碳纳米管可引起A549细胞活性降低、细胞内活性氧ROS过量产生以及谷胱甘肽GSH含量下降, 诱导细胞氧化应激效应; 抗氧化剂N-乙酰半胱氨酸(NAC)抑制多壁碳纳米管诱导的A549细胞内ROS的产生. 多壁碳纳米管处理A549细胞2 h后, 诱发细胞线粒体膜电位下降; 多壁碳纳米管诱导细胞氧化应激的同时伴有适应性应激蛋白HO-1的上调表达. 结果表明, 细胞氧化应激和线粒体膜电位去极化可能是多壁碳纳米管诱导A549细胞毒性效应的重要机制.     

The Anti-Apoptotic Effects of Caspase Inhibitors in Propyl Gallate-Treated Lung Cancer Cells Are Related to Changes in Reactive Oxygen Species and Glutathione Levels

Propyl gallate [3,4,5-trihydroxybenzoic acid propyl ester; PG] exhibits an anti-growth effect in various cells. In this study, the anti-apoptotic effects of various caspase inhibitors were evaluated in PG-treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. Treatment with 800 μM PG inhibited the proliferation and induced the cell death of both Calu-6 and A549 cells at 24 h. Each inhibitor of pan-caspase, caspase-3, caspase-8, and caspase-9 reduced the number of dead and sub-G1 cells in both PG-treated cells at 24 h. PG increased ROS levels, including O₂^∙−, in both lung cancer cell lines at 24 h. Generally, caspase inhibitors appeared to decrease ROS levels in PG-treated lung cancer cells at 24 h and somewhat reduced O₂^∙− levels. PG augmented the number of GSH-depleted Calu-6 and A549 cells at 24 h. Caspase inhibitors did not affect the level of GSH depletion in PG-treated A549 cells but differently and partially altered the depletion level in PG-treated Calu-6 cells. In conclusion, PG exhibits an anti-proliferative effect in Calu-6 and A549 lung cancer cells and induced their cell death. PG-induced lung cancer death was accompanied by increases in ROS levels and GSH depletion. Therefore, the anti-apoptotic effects of caspase inhibitors were, at least in part, related to changes in ROS and GSH levels.     

Oxygen-Deficient Molybdenum Oxide Nanosensitizers for Ultrasound-Enhanced Cancer Metalloimmunotherapy

Oxygen-deficient molybdenum oxide (MoO_X) nanomaterials are prepared as novel nanosensitizers and TME-stimulants for ultrasound (US)-enhanced cancer metalloimmunotherapy. After PEGylation, MoO_X-PEG exhibits efficient capability for US-triggered reactive oxygen species (ROS) generation and glutathione (GSH) depletion. Under US irradiation, MoO_X-PEG generates a massive amount of ROS to induce cancer cell damage and immunogenic cell death (ICD), which can effectively suppress tumor growth. More importantly, MoO_X-PEG itself further stimulates the maturation of dendritic cells (DCs) and triggeres the activation of the cGAS-STING pathway to enhance the immunological effect. Due to the robust ICD induced by SDT and efficient DC maturation stimulated by MoO_X-PEG, the combination treatment of MoO_X-triggered SDT and aCTLA-4 further amplifies antitumor therapy, inhibits cancer metastases, and elicits robust immune responses to effectively defeat abscopal tumors.     

Protective Properties of Novel S‐Acyl‐Glutathione Thioesters Against Ultraviolet‐induced Oxidative Stress

UV‐induced toxicity is characterized by marked oxidative stress, accompanied by the depletion of key cellular antioxidants, particularly glutathione (GSH). Replenishing cellular GSH may represent a means of counteracting UV‐induced toxicity: however, treatment with free GSH is not therapeutically effective due to its unfavorable pharmacokinetic properties. In this study, we show that S‐acyl‐glutathione (acyl‐SG) derivatives, which consist of an acyl chain (of variable length and saturation) linked via a thioester bond to GSH, increase intracellular levels of reduced GSH in primary skin fibroblasts, adenocarcinoma HeLa and neuroblastoma SH‐SY5Y cells. Consistent with this, acyl‐SG derivatives protect against UV‐induced reactive oxygen species (ROS) production and UV‐B/C‐mediated lipid peroxidation and caspase‐3 activation in the analyzed cell lines, with unsaturated thioesters displaying a significantly greater protective effect. Taken together, our findings suggest that acyl‐SG thioesters may be therapeutically effective in the treatment of UV‐related skin disorders and oxidative stress‐mediated conditions in general.     

A fluorescent probe operating under weak acidic conditions for the visualization of HOCl in solid tumors in vivo

Levels of reactive oxygen species(ROS) in cancer cells or in the tumor microenvironment differ noticeably from those in normal cells and cellular microenvironments because ROS play important roles in all aspects of tumor physiology. However, due to the lack of adequate tools, it is difficult to study the relationship between ROS, especially certain types of ROS(e.g., HOCl), and cancer. We report herein an HOCl-specific fluorescent probe, FDOCl-20, containing a thiocarbamide group as a receptor, for the visualization of HOCl in solid tumors in vivo. This probe displays high selectivity and sensitivity to HOCl, and is appropriate for use in acidic conditions, including the tumor microenvironment. Using FDOCl-20 as a tool, we can visualize HOCl in solid tumors in vivo. Importantly, the fluorescent intensity of FDOCl-20 is proportional to tumor volume. Thus, FDOCl-20 is a useful tool to investigate the relationship between HOCl and the physiological processes of tumors.     

Free Radicals as a Double-Edged Sword: The Cancer Preventive and Therapeutic Roles of Curcumin

Free radicals, generally composed of reactive oxygen species (ROS) and reactive nitrogen species (RNS), are generated in the body by various endogenous and exogenous systems. The overproduction of free radicals is known to cause several chronic diseases including cancer. However, increased production of free radicals by chemotherapeutic drugs is also associated with apoptosis in cancer cells, indicating the dual nature of free radicals. Among various natural compounds, curcumin manifests as an antioxidant in normal cells that helps in the prevention of carcinogenesis. It also acts as a prooxidant in cancer cells and is associated with inducing apoptosis. Curcumin quenches free radicals, induces antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), and upregulates antioxidative protein markers–Nrf2 and HO-1 that lead to the suppression of cellular oxidative stress. In cancer cells, curcumin aggressively increases ROS that results in DNA damage and subsequently cancer cell death. It also sensitizes drug-resistant cancer cells and increases the anticancer effects of chemotherapeutic drugs. Thus, curcumin shows beneficial effects in prevention, treatment and chemosensitization of cancer cells. In this review, we will discuss the dual role of free radicals as well as the chemopreventive and chemotherapeutic effects of curcumin and its analogues against cancer.     

Continuous hypoxia attenuates paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line

Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride; PQ), an effective and widely used herbicide, was commercially introduced in 1962. It is reduced by the electron donor NADPH, and then reduced PQ transfers the electrons to molecular oxygen, resulting in the production of reactive oxygen species (ROS), which are related to cellular toxicity. However, the influence of continuous hypoxia on PQ-induced ROS production has not fully been investigated. We evaluated in vitro the protective effect of continuous hypoxia on PQ-induced cytotoxicity in the human carcinogenic alveolar basal epithelial cell line (A549 cells) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and live and dead assay, and by measuring lactate dehydrogenase (LDH) release. To elucidate the mechanism underlying this effect, we monitored the immunofluorescence of intracellular ROS and measured malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. Continuous hypoxia protected the A549 cells from PQ-induced cytotoxicity. Continuous hypoxia for a period of 24 h significantly reduced intracellular ROS, decreased MDA concentration in the supernatant, and normalized SOD and GPx activities. Continuous hypoxia attenuated PQ-induced cell toxicity in A549 cells. This protective effect might be attributable to the suppression of PQ-induced ROS generation.     

Increased melanogenesis is a risk factor for oxidative DNA damage--study on cultured melanocytes and atypical nevus cells

Melanin synthesis is an oxygen-dependent process that acts as a potential source of reactive oxygen species (ROS) inside pigment-forming cells. The synthesis of the lighter variant of melanin, pheomelanin, consumes cysteine and this may limit the capacity of the cellular antioxidative defense. We show that tyrosine-induced melanogenesis in cultured normal human melanocytes (NHM) is accompanied by increased production of ROS and decreased concentration of intracellular glutathione. Clinical atypical (dysplastic) nevi (DN) regularly contain more melanin than do normal melanocytes (MC). We also show that in these cultured DN cells three out of four exhibit elevated synthesis of pheomelanin and this is accompanied by their early senescence. By using various redox-sensitive molecular probes, we demonstrate that cultured DN cells produce significantly more ROS than do normal MC from the same donor. Our experiments employing single-cell gel electrophoresis (comet assay) usually reveal higher fragmentation of DNA in DN cells than in normal MC. Even if in some cases the normal alkaline comet assay shows no differences in DNA fragmentation between DN cells and normal MC, the use of the comet assay with formamidopyrimidine DNA glycosylase can disclose that the DNA of the cultured DN cells harbor more oxidative damage than the DNA of normal MC from the same person.     

Fast Antioxidation Kinetics of Glutathione Intracellularly Monitored by a Dual-Wire Nanosensor

The glutathione (GSH) system is one of the most powerful intracellular antioxidant systems for the elimination of reactive oxygen species (ROS) and maintaining cellular redox homeostasis. However, the rapid kinetics information (at the millisecond to the second level) during the dynamic antioxidation process of the GSH system remains unclear. As such, we specifically developed a novel dual-wire nanosensor (DWNS) that can selectively and synchronously measure the levels of GSH and ROS with high temporal resolution, and applied it to monitor the transient ROS generation as well as the rapid antioxidation process of the GSH system in individual cancer cells. These measurements revealed that the glutathione peroxidase (GPx) in the GSH system is rapidly initiated against ROS burst in a sub-second time scale, but the elimination process is short-lived, ending after a few seconds, while some ROS are still present in the cells. This study is expected to open new perspectives for understanding the GSH antioxidant system and studying some redox imbalance-related physiological.     

A reversible near-infrared fluorescence probe for reactive oxygen species based on Te-rhodamine

We have designed and synthesized a reversible near-infrared (NIR) fluorescence probe, 2-Me TeR, for reactive oxygen species (ROS), utilizing the redox properties of the tellurium (Te) atom. 2-Me TeR is oxidized to fluorescent 2-Me TeOR by various ROS, while the generated 2-Me TeOR is quickly reduced in the presence of glutathione to regenerate 2-Me TeR. This redox-induced reversible NIR-fluorescence response of 2-Me TeR allowed us to detect the endogenous production of ROS and subsequent homeostatic recovery of the intracellular reductive environment in hydrogen peroxide-stimulated HL-60 cells. This probe is expected to be useful for monitoring the dynamics of ROS production continuously in vivo.     

Redox active metal-induced oxidative stress in biological systems

A number of studies performed on biological systems have shown that redox-active metals such as iron and copper as well as other transition metals can undergo redox cycling reactions and produce reactive free radicals termed also reactive oxygen species (ROS) or reactive nitrogen species (RNS). The most representative examples of ROS and RNS are the superoxide anion radical and nitric oxide, respectively, both playing a dual role in biological systems. At low/moderate concentrations of ROS and RNS, they can be involved in many physiological roles such as defense against infectious agents, involvement in a number of cellular signaling pathways and other important biological processes. On the other hand, at high concentrations, ROS and RNS can be important mediators of damage to biomolecules involving DNA, membrane lipids, and proteins. One of the most damaging ROS occurring in biological systems is the hydroxyl radical formed via the decomposition of hydrogen peroxide catalyzed by traces of iron, copper and other metals (the Fenton reaction). The hydroxyl radical is known to react with the DNA molecule, forming 8-OH-Guanine adduct, which is a good biomarker of oxidative stress of an organism and a potential biomarker of carcinogenesis. This review discusses the role of iron and copper in uncontrolled formation of ROS leading to various human diseases such as cancer, cardiovascular disease, and neurological disorders (Alzheimer’s disease and Parkinson’s disease). A discussion is devoted to the various protective antioxidant networks against the deleterious action of free radicals. Metal-chelation therapy, which is a modern pharmacotherapy used to chelate redox-active metals and remove toxic metals from living systems to avoid metal poisoning, is also discussed.     

Antioxidant inhibitors potentiate the cytotoxicity of photodynamic therapy

Photodynamic therapy (PDT) is an increasingly popular anticancer treatment that uses photosensitizer, light and tissue oxygen to generate cytotoxic reactive oxygen species (ROS) within illuminated cells. Acting to counteract ROS-mediated damage are various cellular antioxidant pathways. In this study, we combined PDT with specific antioxidant inhibitors to potentiate PDT cytotoxicity in MCF-7 cancer cells. We used disulphonated aluminium phthalocyanine photosensitizer plus various combinations of the antioxidant inhibitors: diethyl-dithiocarbamate (DDC, a Cu/Zn-SOD inhibitor), 2-methoxyestradiol (2-ME, a Mn-SOD inhibitor), l-buthionine sulfoximine (BSO, a glutathione synthesis inhibitor) and 3-amino-1,2,4-triazole (3-AT, a catalase inhibitor). BSO, singly or in combination with other antioxidant inhibitors, significantly potentiated PDT cytotoxicity, corresponding with increased ROS levels and apoptosis. The greatest potentiation of cell death over PDT alone was seen when cells were preincubated for 24 h with 300 μM BSO plus 10 mM 3-AT (1.62-fold potentiation) or 300 μM BSO plus 1 μM 2-ME (1.52-fold), or with a combination of all four inhibitors (300 μM BSO, 10 mM 3-AT, 1 μM 2-ME and 10 μM DDC: 1.4-fold). As many of these inhibitors have already been clinically tested, this work facilitates future in vivo studies.     

Photodynamic treatment with fractionated light decreases production of reactive oxygen species and cytotoxicity in vitro via regeneration of glutathione

Photodynamic therapy removes unwanted or harmful cells by overproduction of reactive oxygen species (ROS). Fractionated light delivery in photodynamic therapy may enhance the photodynamic effect in tumor areas with insufficient blood supply by enabling the reoxygenation of the treated area. This study addresses the outcome of fractionated irradiation in an in vitro photodynamic treatment (PDT) system, where deoxygenation can be neglected. Our results show that fractionated irradiation with light/dark intervals of 45/60 s decreases ROS production and cytotoxicity of PDT. This effect can be reversed by addition of 1,3-bis-(2-chlorethyl)-1-nitrosurea (BCNU), an inhibitor of the glutathione reductase. We suggest that the dark intervals during irradiation allow the glutathione reductase to regenerate reduced glutathione (GSH), thereby rendering cells less susceptible to ROS produced by PDT compared with continuous irradiation. Our results could be of particular clinical importance for photodynamic therapy applied to well-oxygenated tumors.     

天然水体中腐殖质的光化学研究进展

本文综述了近年来天然水体中腐殖质的光化学研究进展,重点讨论腐殖质以及相关活性氧的光化学反应机理,并分析了影响腐殖质光解的主要因素,最后,针对水体腐殖质光化学研究中面临的问题,提出了今后的重点研究方向.     

Nanotechnology for Electroanalytical Biosensors of Reactive Oxygen and Nitrogen Species

Over the past several decades, nanotechnology has contributed to the progress of biomedicine, biomarker discovery, and the development of highly sensitive electroanalytical / electrochemical biosensors for in vitro and in vivo monitoring, and quantification of oxidative and nitrosative stress markers like reactive oxygen species (ROS) and reactive nitrogen species (RNS). A major source of ROS and RNS is oxidative stress in cells, which can cause many human diseases, including cancer. Therefore, the detection of local concentrations of ROS (e. g. superoxide anion radical; O₂^•−) and RNS (e. g. nitric oxide radical; NO^• and its metabolites) released from biological systems is increasingly important and needs a sophisticated detection strategy to monitor ROS and RNS in vitro and in vivo. In this review, we discuss the nanomaterials‐based ROS and RNS biosensors utilizing electrochemical techniques with emphasis on their biomedical applications.     

Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis

A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.