Anionic phospholipid-induced regulation of reactive oxygen species production by human cytochrome P450 2E1

We suggest that the cytochrome P450 2E1 (CYP2E1)-induced formation of reactive oxygen species (ROS) can be regulated by anionic phospholipids and the presence of the N-terminal region of the enzyme. When the content of cardiolipin (CL) in membranes at the expense of phosphatidylcholine matrix was increased, the ROS produced by recombinant human CYP2E1 was decreased as a function of CL concentration. On the contrary, the N-terminally truncated CYP2E1 had a decreased effect on the lipid-induced reduction of ROS formation. These results suggest that specific phospholipids can regulate the function of CYP2E1 by interaction with the enzyme including the N-terminal region(s).     

Inactivated Sendai virus strain Tianjin induces apoptosis and autophagy through reactive oxygen species production in osteosarcoma MG-63 cells

Sendai virus strain Tianjin, a novel genotype of Sendai virus, has been proven to possess potent antitumor effect on certain cancer cell types although inactivated by ultraviolet (UV). This study was carried out to investigate the in vitro anticancer properties of UV-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human osteosarcoma cells and the underlying molecular mechanism. Our studies demonstrated UV-Tianjin significantly inhibited the viability of human osteosarcoma cell lines and triggered apoptosis through activation of both extrinsic and intrinsic pathways in MG-63 cells. Meanwhile, autophagy occurred in UV-Tianjin-treated cells. Blockade of autophagy with 3-methyladenine remarkably attenuated the inhibition of cell proliferation by UV-Tianjin, suggesting that UV-Tianjin-induced autophagy may be contributing to cell death. Furthermore, UV-Tianjin induced reactive oxygen species (ROS) production, which was involved in the execution of MG-63 cell apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-L-cysteine, a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of apoptosis promoted autophagy, whereas suppression of autophagy attenuated apoptosis. Our results suggest that UV-Tianjin triggers apoptosis and autophagic cell death via generation of the ROS in MG-63 cells, which might provide important insights into the effectiveness of novel strategies for osteosarcoma therapy.     

Ethanol increases mitochondrial cytochrome P450 2E1 in mouse liver and rat hepatocytes

Enhanced hepatic levels of cytochrome P450 2E1 (CYP2E1) may play a key role in the pathogenesis of some liver diseases because CYP2E1 represents a significant source of reactive oxygen species. Although a large fraction of CYP2E1 is located in the endoplasmic reticulum, CYP2E1 is also present in mitochondria. In this study, we asked whether ethanol, a known inducer of microsomal CYP2E1, could also increase CYP2E1 within mitochondria. Our findings indicated that ethanol increased microsomal and mitochondrial CYP2E1 in cultured rat hepatocytes and in the liver of lean mice. This was associated with decreased levels of glutathione, possibly reflecting increased oxidative stress. In contrast, in leptin-deficient obese mice, ethanol administration did not increase mitochondrial CYP2E1, nor it depleted mitochondrial glutathione, suggesting that leptin deficiency hampers mitochondrial targeting of CYP2E1. Thus, ethanol intoxication increases CYP2E1 not only in the endoplasmic reticulum but also in mitochondria, thus favouring oxidative stress in these compartments.     

光果甘草毛状根培养过程中对活性氧清除能力和总黄酮含量的变化

采用化学发光法, 分析了光果甘草(Glycyrrhiza glabra L.)毛状根培养过程中对3种活性氧(ROS: O-2(÷)、HO·和H-2O-2)清除能力的动态变化, 并测定了培养过程中总黄酮含量的动态变化. 实验结果表明, 毛状根在对数生长期(20～28 d)对3种ROS都有很强的清除能力,在生长停滞期(29～40 d)对HO·和H-2O-2的清除能力仍维持较高的水平,而对O-2(÷)的清除能力随培养时间的延长逐渐下降.总黄酮含量在对数生长期呈现增加的趋势,至31 d时达到最高含量(0.78%),随培养时间的延长含量逐渐降低.     

Mitochondrial Disease-related Mutation G167P in Cytochrome b of Rhodobacter capsulatus Cytochrome bc1 (S151P in Human) Affects the Equilibrium Distribution of [2Fe-2S] Cluster and Generation of Superoxide

Cytochrome bc₁ is one of the key enzymes of many bioenergetic systems. Its operation involves a large scale movement of a head domain of iron-sulfur protein (ISP-HD), which functionally connects the catalytic quinol oxidation Q_o site in cytochrome b with cytochrome c₁. The Q_o site under certain conditions can generate reactive oxygen species in the reaction scheme depending on the actual position of ISP-HD in respect to the Q_o site. Here, using a bacterial system, we show that mutation G167P in cytochrome b shifts the equilibrium distribution of ISP-HD toward positions remote from the Q_o site. This renders cytochrome bc₁ non-functional in vivo. This effect is remediated by addition of alanine insertions (1Ala and 2Ala) in the neck region of the ISP subunit. These insertions, which on their own shift the equilibrium distribution of ISP-HD in the opposite direction (i.e. toward the Q_o site), also act in this manner in the presence of G167P. Changes in the equilibrium distribution of ISP-HD in G167P lead to an increased propensity of cytochrome bc₁ to generate superoxide, which becomes evident when the concentration of quinone increases. This result corroborates the recently proposed model in which “semireverse” electron transfer back to the Q_o site, occurring when ISP-HD is remote from the site, favors reactive oxygen species production. G167P suggests possible molecular effects of S151P (corresponding in sequence to G167P) identified as a mitochondrial disease-related mutation in human cytochrome b. These effects may be valid for other human mutations that change the equilibrium distribution of ISP-HD in a manner similar to G167P.     

Melamine causes apoptosis of rat kidney epithelial cell line (NRK-52e cells) via excessive intracellular ROS (reactive oxygen species) and the activation of p38 MAPK pathway

There was an outbreak of urinary stones associated with consumption of melamine-tainted milk products in 2008 in China, leading to serious illness of many infants and even death. We have recently demonstrated that melamine causes oxidative damage on the NRK (normal rat kidney)-52e cells. The objective of this study was to explore the cellular signalling pathway that mediates the cell apoptosis induced by melamine in the NRK-52e cells. Fluorescence microscope showed that melamine enhanced intracellular ROS (reactive oxygen species) levels of the NRK-52e cells. AO/EB (acridine orange/ethidium bromide) staining and flow cytometry revealed that melamine increased apoptotic and necrotic percentages of the NRK-52e cells in a dose-dependent manner. Notably, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assays and flow cytometry displayed that SB203580, an inhibitor for p38 MAPK (mitogen-activated protein kinase) pathway, increased the proliferation of the NRK-52e cells and reduced the apoptotic and necrotic percentages of the NRK-52e cells. Western blots further demonstrated that p38 phosphorylation was activated by melamine in the NRK-52e cells and inhibitor SB203580 blocked the increase of p38 phosphorylation induced by melamine. Together, these results suggested that melamine causes apoptosis of the NRK-52e cells via excessive intracellular ROS and the activation of p38 MAPK pathway. This study thus offers a novel insight into molecular mechanisms by which melamine has adverse cytotoxicity on renal tubular epithelial cells.     

Zinc induces distinct changes in the metabolism of reactive oxygen and nitrogen species (ROS and RNS) in the roots of two Brassica species with different sensitivity to zinc stress

Background and Aims Zinc (Zn) is an essential micronutrient naturally present in soils, but anthropogenic activities can lead to accumulation in the environment and resulting damage to plants. Heavy metals such as Zn can induce oxidative stress and the generation of reactive oxygen and nitrogen species (ROS and RNS), which can reduce growth and yield in crop plants. This study assesses the interplay of these two families of molecules in order to evaluate the responses in roots of two Brassica species under high concentrations of Zn.Methods Nine-day-old hydroponically grown Brassica juncea (Indian mustard) and B. napus (oilseed rape) seedlings were treated with ZnSO₄ (0, 50, 150 and 300 µm) for 7 d. Stress intensity was assessed through analyses of cell wall damage and cell viability. Biochemical and cellular techniques were used to measure key components of the metabolism of ROS and RNS including lipid peroxidation, enzymatic antioxidants, protein nitration and content of superoxide radical (O2·−), nitric oxide (NO) and peroxynitrite (ONOO⁻).Key Results Analysis of morphological root damage and alterations of microelement homeostasis indicate that B. juncea is more tolerant to Zn stress than B. napus. ROS and RNS parameters suggest that the oxidative components are predominant compared with the nitrosative components in the root system of both species.Conclusions The results indicate a clear relationship between ROS and RNS metabolism as a mechanism of response against stress caused by an excess of Zn. The oxidative stress components seem to be more dominant than the elements of the nitrosative stress in the root system of these two Brassica species.     

Fibrotic response induced by angiotensin-II requires NAD(P)H oxidase-induced reactive oxygen species (ROS) in skeletal muscle cells

Fibrotic disorders are typified by excessive connective tissue and extracellular matrix (ECM) deposition that precludes normal healing processes in different tissues. Angiotensin-II (Ang-II) is involved in the fibrotic response. Several muscular dystrophies are characterized by extensive fibrosis. However, the exact role of Ang-II in skeletal muscle fibrosis is unknown. Here we show that myoblasts responded to Ang-II by increasing protein levels of connective tissue growth factor (CTGF/CCN2), collagen-III and fibronectin. These Ang-II-induced pro-fibrotic effects were mediated by AT-1 receptors. Remarkably, Ang-II induced reactive oxygen species (ROS) via a NAD(P)H oxidase-dependent mechanism, as shown by inhibition of ROS production via the NAD(P)H oxidase inhibitors diphenylene iodonium (DPI) and apocynin. This increase in ROS is critical for Ang-II-induced fibrotic effects, as indicated by the decrease in Ang-II-induced CTGF and fibronectin levels by DPI and apocynin. We also show that Ang-II-induced ROS production and fibrosis require PKC activity as indicated by the generic PKC inhibitor chelerythrine.These results strongly suggest that the fibrotic response induced by Ang-II is mediated by AT-1 receptor and requires NAD(P)H-induced ROS in skeletal muscle cells.     

Role of reactive oxygen species and NADPH-oxidase in the development of rat cerebellum

Experimental evidence suggests that reactive oxygen species (ROS) could participate in the regulation of some physiological conditions. In the nervous system, ROS have been suggested to act as signaling molecules involved in several developmental processes including cell differentiation, proliferation and programmed of cell death. Although ROS can be generated by several sources, it has been suggested that NADPH oxidase (NOX) could be critical in the production of ROS acting as a signal in some of these events. It has been reported that ROS production by NOX enzymes participate in neuronal maturation and differentiation during brain development. In the present study, we found that during rat cerebellar development there was a differential ROS generation at different ages and areas of the cerebellum. We also found a differential expression of NOX homologues during rat cerebellar development. When we treated developing rats with an antioxidant or with apocynin, an inhibitor of NOX, we found a marked decrease of the ROS levels in all the cerebellar layers at all the ages tested. Both treatments also induced a significant change in the cerebellar foliation as well as an alteration in motor behavior. These results suggest that both ROS and NOX have a critical role during cerebellar development.     

Differential effects of glucose deprivation on the cellular sensitivity towards photodynamic treatment-based production of reactive oxygen species and apoptosis-induction

Photodynamic treatment (PDT) employs a photosensitizer and the light-induced formation of reactive oxygen species--antagonized by cellular antioxidant systems--for the removal of harmful cells. This study addresses the effect of altered carbohydrate metabolism on the cellular antioxidant glutathione system, and the subsequent responses to PDT. It is shown that glucose-deprivation of 18 h prior to PDT causes a reduced level of intracellular glutathione and an increased cytotoxicity of PDT. These effects can be mimicked by inhibitors of glutathione synthesis (buthionine-sulfoximine) or its regeneration (1,3-bis-(2-chlorethyl)-1-nitrosourea). Inhibited glutathione metabolism shifts the apoptotic window to lower fluences, while glucose deprivation abolishes apoptosis as a result of ATP deficiency. Our results prove evidence for manipulation of the outcome of PDT through internal metabolic pathways.     

Phospholipase D in the signaling networks of plant response to abscisic acid and reactive oxygen species

Phospholipase D (PLD) has been implicated in different cellular processes in plant growth, development, and stress responses. Recent results have provided insights into the molecular mechanism by which PLD and its lipid product phosphatidic acid (PA) participate in cell signaling. Effector proteins that have been identified for PLD and PA in plants include a heterotrimeric G protein, protein phosphatase, and protein kinase. Evidence has been presented for a direct link from a PLD, PA, to a target protein in specific physiological processes. PLD and PA play multiple roles in the signaling networks of plant response to abscisic acid and reactive oxygen species.     

Bimodal actions of reactive oxygen species in the differentiation and bone-resorbing functions of osteoclasts

In order to demonstrate that cellular redox status undergoes decreased reduction during osteoclast differentiation and further decreased reduction during osteoclastic bone resorption, we analyzed gamma-glutamylcysteinyl synthetase activity, a glutathione synthesis rate-limiting enzyme, and total glutathione and thiol groups. Moderate and severe redox shifts towards a more oxidizing environment induced gradual increases and decreases in osteoclastogenesis. Moreover, while severe glutathione depletion inhibited bone resorption, moderate glutathione repletion enhanced bone resorption. In summary, our observations suggest that there is a threshold for redox status, representing biphasic patterns in osteoclast differentiation and function.     

The role of aryl hydrocarbon receptor and the reactive oxygen species in the modulation of glutathione transferase by heavy metals in murine hepatoma cell lines

Glutathione transferase (GST) is a phase II detoxifying enzyme that plays a protective mechanism against oxidizing substances and toxic contaminants. Among these contaminants, heavy metals and polycyclic and halogenated aromatic hydrocarbons (PHAHs) have been shown to exert their toxic effects through the modulation of detoxifying enzymes, including the GSTs. Recently, we showed that heavy metals particularly Hg2+, Pb2+, and Cu2+ modulate the expression of phase II detoxifying enzymes such as NAD(P)H:quinone oxidoreductase 1 and Gsta1 in a concentration- and time-dependent manner. However, the effect of heavy metals and their potential interactions with aryl hydrocarbon receptor (AhR) ligands, PHAHs, on total Gst activity is still unknown. In the current study, we have investigated the effects of Hg2+, Pb2+, and Cu2+ in the absence and presence of four AhR ligands on the total Gst activity and reactive oxygen species (ROS) production in wild-type and AhR-deficient Hepa 1c1c7 cells. Our results showed that Hg2+ and Cu2+, but not Pb2+, significantly induced Gst activity in wild-type cells, whereas all metals induced the Gst activity in AhR-deficient cells. The induction of Gst activity by heavy metals was strongly correlated with an increase in the ROS production in wild-type, but not in AhR-deficient cells. Co-administration of heavy metals with AhR ligands differentially modulated Gst activity, in that co-exposure to Hg2+ plus AhR ligands could be beneficial in protecting against cytotoxicity as demonstrated by the increase in Gst activity with a proportional decrease in ROS production. Whereas co-exposure to Cu2+ plus AhR ligands was more toxic in that a decrease in Gst activity and an increase in oxidative stress of the cell were observed. We concluded that heavy metals differentially modulate the Gst activity through oxidative stress- and AhR-mediated mechanisms.     

Oxidases and peroxidases in cardiovascular and lung disease: new concepts in reactive oxygen species signaling

Reactive oxygen species (ROS) are involved in numerous physiological and pathophysiological responses. Increasing evidence implicates ROS as signaling molecules involved in the propagation of cellular pathways. The NADPH oxidase (Nox) family of enzymes is a major source of ROS in the cell and has been related to the progression of many diseases and even environmental toxicity. The complexity of this family's effects on cellular processes stems from the fact that there are seven members, each with unique tissue distribution, cellular localization, and expression. Nox proteins also differ in activation mechanisms and the major ROS detected as their product. To add to this complexity, mounting evidence suggests that other cellular oxidases or their products may be involved in Nox regulation. The overall redox and metabolic status of the cell, specifically the mitochondria, also has implications on ROS signaling. Signaling of such molecules as electrophilic fatty acids has an impact on many redox-sensitive pathologies and thus, as anti-inflammatory molecules, contributes to the complexity of ROS regulation. This review is based on the proceedings of a recent international Oxidase Signaling Symposium at the University of Pittsburgh's Vascular Medicine Institute and Department of Pharmacology and Chemical Biology and encompasses further interaction and discussion among the presenters.     

The impact of reactive oxygen species and genetic mitochondrial mutations in Parkinson's disease

The exact pathogenesis of Parkinson's disease (PD) is still unknown and proper mechanisms that correspond to the disease remain unidentified. It is understood that PD is age-related; as age increases, the chance of onset responds accordingly. Although there are no current means of curing PD, the understanding of reactive oxygen species (ROS) provides significant insight to possible treatments. Complex I deficiencies of the respiratory chain account for the majority of unfavorable neural apoptosis generation in PD. Dopaminergic neurons are severely damaged as a result of the deficiency. Symptoms such as inhibited cognitive ability and loss of smooth motor function are the results of such impairment. The genetic mutations of Parkinson's related proteins such as PINK1 and LRRK2 contribute to mitochondrial dysfunction which precedes ROS formation. Various pathways are inhibited by these mutations, and inevitably causing neural cell damage. Antioxidants are known to negate the damaging effects of free radical overexpression. This paper expands on the specific impact of mitochondrial genetic change and production of free radicals as well as its correlation to the neurodegeneration in Parkinson's disease.