Metabolic Depression and Increased Reactive Oxygen Species Production by Isolated Mitochondria at Moderately Lower Temperatures

Temperature (T) reduction increases lifespan, but the mechanisms are not understood. Because reactive oxygen species (ROS) contribute to aging, we hypothesized that lowering T might decrease mitochondrial ROS production. We measured respiratory response and ROS production in isolated mitochondria at 32, 35, and 37 °C. Lowering T decreased the rates of resting (state 4) and phosphorylating (state 3) respiration phases. Surprisingly, this respiratory slowdown was associated with an increase of ROS production and hydrogen peroxide release and with elevation of the mitochondrial membrane potential, ΔΨ_m. We also found that at lower T mitochondria produced more carbon-centered lipid radicals, a species known to activate uncoupling proteins. These data indicate that reduced mitochondrial ROS production is not one of the mechanisms mediating lifespan extension at lower T. They suggest instead that increased ROS leakage may mediate mitochondrial responses to hypothermia.     

Overexpression of Rice Ferrochelatase I and II Leads to Increased Susceptibility to Oxyfluorfen Herbicide in Transgenic Rice

Protoporphyrin IX is a photosensitizer and a causative agent of rice membrane lipid peroxidation in plant cells. Protoporphyrinogen IX oxidase (PPO) is the molecular target of PPO-inhibiting herbicides, which trigger a massive increase in protoporphyrin IX. Thus, any possible method to decrease the levels of protoporphyrin IX upon challenge with PPO-inhibiting herbicides could be employed to generate plants resistant to such herbicides. We generated transgenic rice plants overexpressing rice ferrochelatase isogenes encoding ferrochelatase enzymes, which convert protoporphyrin IX into protoheme, to see whether the transgenic plants have phenotypes resistant to PPO-inhibiting herbicides. The resulting transgenic rice plants were all susceptible to oxyfluorfen (a diphenyl-ether-type PPO-inhibiting herbicide), as judged by cellular damage with respect to cellular leakage, chlorophyll loss, and lipid peroxidation. In particular, the transgenic plants expressing rice ferrochelatase II without its plastid targeting sequence showed higher transgene expression and oxyfluorfen susceptibility than lines expressing the intact ferrochelatase II. Possible susceptibility mechanisms to oxyfluorfen herbicide in the transgenic rice plants are discussed.     

Mitochondria of Isolated Plant Cells (Acer pseudoplatanus L.): II. Copper Deficiency Effects on Cytochrome C Oxidase and Oxygen Uptake

The effects of copper deficiency on cell culture growth, cell respiration, mitochondrial oxidative properties, and electron transport chain have been studied with suspension-cultured sycamore cells (Acer pseudoplatanus L.). Within the range of the copper concentration studied (0.1-25 μg/1 of culture medium), the mean rate of cell division is independent of copper concentration. An initial copper concentration lower than 2 μg/1 limited the maximum density of population reached at the stationary phase of growth.     

Biochemical, Histological, and Memory Impairment Effects of Chronic Copper Toxicity: A Model for Non-Wilsonian Brain Copper Toxicosis in Wistar Rat

Animal models of copper toxicosis rarely exhibit neurological impairments and increased brain copper accumulation impeding the development of novel therapeutic approaches to treat neurodegenerative diseases having high brain Cu content. The aim of this study was to investigate the effects of intraperitoneally injected copper lactate (0.15 mg Cu/100 g body weight) daily for 90 days on copper and zinc levels in the liver and hippocampus, on biochemical parameters, and on neurobehavioral functions (by Morris water maze) of male Wistar rats. Copper-administered animals exhibited significantly decreased serum acetylcholinesterase (AChE) activity and impaired neuromuscular coordination and spatial memory compared to control rats. Copper-intoxicated rats showed significant increase in liver and hippocampus copper content (99.1 and 73 % increase, respectively), 40.7 % reduction in hepatic zinc content, and interestingly, 77.1 % increase in hippocampus zinc content with concomitant increase in copper and zinc levels in serum and urine compared to control rats. Massive grade 4 copper depositions and grade 1 copper-associated protein in hepatocytes of copper-intoxicated rats were substantiated by rhodanine and orcein stains, respectively. Copper-intoxicated rats demonstrated swelling and increase in the number of astrocytes and copper deposition in the choroid plexus, with degenerated neurons showing pyknotic nuclei and dense eosinophilic cytoplasm. In conclusion, the present study shows the first evidence in vivo that chronic copper toxicity causes impaired spatial memory and neuromuscular coordination, swelling of astrocytes, decreased serum AChE activity, copper deposition in the choroid plexus, neuronal degeneration, and augmented levels of copper and zinc in the hippocampus of male Wistar rats.     

In Vitro Effect of Copper Chloride Exposure on Reactive Oxygen Species Generation and Respiratory Chain Complex Activities of Mitochondria Isolated from Broiler Liver

This study is to examine if Cu(2+) can act directly on mitochondria or indirectly by producing reactive oxygen species (ROS), isolated broiler hepatic mitochondria were exposed to different concentrations of Cu(2+) (10, 30, 50?μM). Respiratory chain complex activities, ROS generation, respiratory control ratio (RCR) and mitochondrial membrane potential were investigated. Dose-dependent inhibition of respiratory chain complexes and induction of ROS were observed, which coincided with decreasing RCR both with glutamate?+?malate or succinate. Further investigation indicated that the membrane potential determined by rhodamine 123 release decreased after CuCl(2) exposure at 30 and 50?μM. In addition, the effects of the antioxidants NAC (200?μM) and GSH (200?μM) were studied at 50?μM Cu(2+). The results indicate that Cu can induce mitochondrial dysfunction in excessive dose and the effect of Cu(2+) exposure on respiratory chain is not site-specific, and antioxidants can protect the mitochondrial function by reducing the formation of free radicals.     

Effect of Synthetic Detergents on the Swelling and the ATPase of Mitochondria Isolated from Rat Liver

A study was made of the effects of various types of detergents on the swelling of isolated mitochondria and on mitochondrial ATPases which are activated by Mg or DNP respectively. The rate of swelling was measured in the Beckman spectrophotometer by following the decrease in turbidity of dilute suspensions of these organelles. It was found that non-ionic detergents containing a nonyl phenoxy side chain or anionic detergents caused swelling of the mitochondria and activation of Mg-ATPase. On the other hand, cationic detergents promoted the clumping of mitochondria and did not activate Mg-ATPase. DNP-ATPase was inhibited by all of the detergents tested. It would appear from these observations that the inhibition of DNP-ATPase is not related to a gross change in the morphology of the organelles; in contrast, the activation of Mg-ATPase definitely is correlated with swelling of the isolated mitochondria. These data also suggest that the ionic detergents combine with charged sites on the protein moiety of the lipoprotein in the mitochondrial surface, whereas the non-ionic detergents form inclusion compounds with the lipide moiety, thereby altering the mitochondrial structure and permeability.     

Carbonic Anhydrase Inhibitors: Metal Complexes of a Sulfanilamide Derived Schiff base and their Interaction with Isozymes I,II and IV

Metal complexes of aromatic/heterocyclic sulfonamides act as stronger inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) as compared to the uncomplexed sulfonamides from which they are derived. Here we report the synthesis and inhibition studies against the physiologically relevant isozymes CA I, CA II and CA IV, of a series of metal complexes (Co(II), Ni(II) and Cu(II) derivatives) of a Schiff-base ligand, obtained from sulfanilamide and salicylaldehyde. The best activity was observed for the Cu(II) and Co(II) complexes, against CA II and CA IV, for which inhibition constants in the range of 15-39 and 72-108 nM, respectively, were seen. The enhanced efficacy in inhibiting the enzyme may be due to a dual mechanism of action of the metal complexes, which interact with CA both by means of the sulfonamide moieties as well as the metal ions present in their molecule.     

Complexation and Toxicity of Copper in Higher Plants. I. Characterization of Copper Accumulation,Speciation, and Toxicity in Crassula helmsii as a New Copper Accumulator

The amphibious water plant Crassula helmsii is an invasive copper (Cu)-tolerant neophyte in Europe. It now turned out to accumulate Cu up to more than 9,000 ppm in its shoots at 10 μm (=0.6 ppm) Cu²⁺ in the nutrient solution, indicating that it is a Cu hyperaccumulator. We investigated uptake, binding environment, and toxicity of Cu in this plant under emerged and submerged conditions. Extended x-ray absorption fine structure measurements on frozen-hydrated samples revealed that Cu was bound almost exclusively by oxygen ligands, likely organic acids, and not any sulfur ligands. Despite significant differences in photosynthesis biochemistry and biophysics between emerged and submerged plants, no differences in Cu ligands were found. While measurements of tissue pH confirmed the diurnal acid cycle typical for Crassulacean acid metabolism, Δ¹³C measurements showed values typical for regular C3 photosynthesis. Cu-induced inhibition of photosynthesis mainly affected the photosystem II (PSII) reaction center, but with some unusual features. Most obviously, the degree of light saturation of electron transport increased during Cu stress, while maximal dark-adapted PSII quantum yield did not change and light-adapted quantum yield of PSII photochemistry decreased particularly in the first 50 s after onset of actinic irradiance. This combination of changes, which were strongest in submerged cultures, shows a decreasing number of functional reaction centers relative to the antenna in a system with high antenna connectivity. Nonphotochemical quenching, in contrast, was modified by Cu mainly in emerged cultures. Pigment concentrations in stressed plants strongly decreased, but no changes in their ratios occurred, indicating that cells either survived intact or died and bleached quickly.Heavy metals such as cadmium (Cd), copper (Cu), manganese, nickel (Ni), and zinc (Zn) are well known to be essential microelements for the life of plants (for Cd, see Lane and Morel, 2000). On the other hand, elevated concentrations of these metals induce inhibition of various processes in plant metabolism (for review, see Prasad and Hagemeyer, 1999; Küpper and Kroneck, 2005). Cu can occur in very high concentrations that are detrimental or even lethal to most plants. It is widely used as a pesticide in agriculture, and field runoff may easily reach concentrations of several micromolar (Gallagher et al., 2001). Photosynthetic reactions, both photochemical and biochemical ones, belong to the most important sites of inhibition by many heavy metals and in particular Cu. In the thylakoids, PSII has frequently been identified to be the main target. The exact location of its damage, however, strongly depends on the irradiance conditions, as shown originally by Cedeno-Maldonado et al. (1972) and later by Küpper et al. (1996b, 1998, 2002). The latter authors found that in low irradiance including a dark phase, the inhibition of PSII is largely due to the impairment of the correct function of the light-harvesting antenna; this mechanism was termed “shade reaction.” It results from the substitution by heavy metals of the Mg²⁺ ion in the chlorophyll (Chl) molecules of the light-harvesting complex II. In high irradiance, direct damage to the PSII reaction center (RC) occurs instead, which most likely involves insertion of Cu²⁺ into the Pheo a of the PSII RC. This was named “sun reaction” (Küpper et al., 1996b, 1998, 2002). Also, oxidative stress has often been described as a result of Cu stress; recent data have shown that in photosynthetic organisms, it is mainly a consequence of an inhibition of the photosynthetic light reactions (Rocchetta and Küpper, 2009).Plants developed a number of strategies to resist the toxicity of heavy metals, as reviewed by Cobbett and Goldsbrough (2002) and Küpper and Kroneck (2005). Such strategies include efflux pumps, sequestration in cells and intracellular compartments where metals do least harm, and binding of heavy metals inside the cells by strong ligands like phytochelatins or free amino acids. A majority of the heavy metal-resistant plants, called “excluders,” prevent the accumulation of heavy metals inside their tissues (Baker, 1981). Other resistant plants actively take up heavy metals, translocate them into the shoot, and sequester them to certain parts of the plant, where they are stored in a harmless state. These plants, which accumulate up to several percent of heavy metals in the dry mass of their aboveground parts, are called “hyperaccumulators” (Brooks et al., 1977). In their natural habitats, metal-rich soils in many parts of the world, this type of heavy metal accumulation serves as a defense against pathogens and herbivores (Boyd and Martens, 1994; Martens and Boyd, 1994; Boyd et al., 2002; Hanson et al., 2003; Jhee et al., 2005). They can now be used for the decontamination (“phytoremediation”) of anthropogenically heavy metal-contaminated soils and in some cases also for the commercial extraction (“phytomining”) of high-value metals (mainly Ni) from metal-rich soils (Baker et al., 1994; McGrath and Zhao, 2003; Chaney et al., 2005).The mechanisms by which hyperaccumulator plants accumulate the enormous amounts of heavy metals in their shoots and prevent phytotoxicity of these metals have been the subject of many studies. Nevertheless, many of these mechanisms are still under debate (Pollard et al., 2002; Küpper and Kroneck, 2005), and a short overview is given in our companion article (Mijovilovich et al., 2009) on Cu in the Cd/Zn model hyperaccumulator plant Thlaspi caerulescens. Studies of arsenic, Cd, Ni, and Zn binding in hyperaccumulators (Krämer et al., 1996; Sagner et al., 1998; Salt et al., 1999, Wang et al., 2002; Küpper et al., 2004) indicated that in such plants most of the metals are coordinated by organic acids, which are commonly found in plant vacuoles. Nonaccumulator plants, in contrast, are well known to bind heavy metals by strong sulfur ligands such as phytochelatins (mainly for Cd) and metallothioneins (mainly for Cu), as reviewed by Cobbett and Goldsbrough (2002).While hundreds of species have been found to hyperaccumulate Ni and about two dozen to hyperaccumulate Zn, true Cu hyperaccumulation in the sense of reaching thousands of ppm in the shoot dry weight has rarely been confirmed. Most species reported to be Cu hyperaccumulators before were later found to be false positives due to Cu adsorption on the leaf surface, et cetera; actually, none of the species recently revisited had a bioaccumulation factor larger than 1, which is commonly regarded as a necessary prerequisite of true hyperaccumulation (Faucon et al., 2007). But it is important in terms of the general understanding of metal metabolism in plants to identify how plants can cope with Cu toxicity other than excluding it from their metabolism and how far the mechanisms of Cu detoxification and Cu stress differ in Cu-resistant and -accumulating plants from Cu excluders and Cu-sensitive plants. Such questions are important also for breeding better Cd/Zn hyperaccumulators, since such plants (e.g. T. caerulescens) turned out to be Cu sensitive, limiting their phytoremediation potential on soils with mixed contamination (Walker and Bernal, 2004). We now analyzed Cu accumulation and Cu stress in a so far not well-characterized species, the amphibious Crassula helmsii, an aggressively invasive plant in Europe (Küpper et al., 1996a). We chose this plant because in a previous study it had turned out to be much more Cu resistant than all other investigated species (Küpper et al., 1996b), but more in summer than in winter. Moreover, preliminary experiments indicated that under high temperatures and salinity, C. helmsii switches to circadian acid metabolism (CAM), which might cause its elevated Cu resistance in summer due to the enhanced availability of malate as a Cu ligand. CAM metabolism was first reported for C. helmsii by Newman and Raven (1995).In this study, we investigated physiological mechanisms of Cu-induced inhibition of photosynthesis, Crassulacean acid metabolism induction, and Cu accumulation and complexation in C. helmsii. The most important method for our investigations of Cu stress was the two-dimensional (imaging) and spectrally resolved microscopic in vivo measurement of the transients of Chl variable fluorescence in the fluorescence kinetic microscope (FKM; Küpper et al., 2000a, 2007a). Cu ligands were investigated via EXAFS (

小鼠肝再生过程中ROS及线粒体代谢变化规律的研究

目的:肝脏是维持人体发挥功能的重要器官,同时肝脏再生能力十分强大。本文通过部分肝切除术后小鼠肝再生模型,观察肝再生过程中氧化应激及线粒体代谢变化规律,以期为将来的调控肝再生提供新的干预靶点。方法:选择雄性健康体重均匀的Balb/c小鼠,采用经典70%肝切除模型,随机分为假手术对照组(Sham组)以及70%肝切除组(70%PH组)。肝切除术后6 h、1d、2 d、3 d、5 d、7 d不同时间点取肝组织,制备冰冻切片检测活性氧(ROS)水平,Western blot分别检测细胞增殖相关蛋白PCNA、Cyclin D1;氧化应激相关蛋白SOD1、SOD2、CAT、GPX1;以及线粒体代谢相关蛋白PGC-1α、Nrf1、TFAM、Drp1、Fis1、Mfn1、Mfn2、OPA1的表达并分析其变化规律。结果:70%肝切除术后小鼠肝脏增长迅速,细胞增殖关键蛋白PCNA和Cyclin D1表达显著增加;在此过程中细胞ROS水平呈现先升高后降低的变化,细胞主要抗氧化酶SOD1、SOD2、CAT、Gpx1与ROS相一致出现先升高后降低的变化。线粒体生物合成调控因子PGC-1α、Nrf1、TFAM呈现先降低后升高的趋势,而线粒体分裂蛋白Drp1和Fis1呈现先降低后显著升高的趋势,线粒体融合相关蛋白Mfn1、Mfn2和OPA1总体为先降低后恢复至正常水平。结论:在小鼠70%肝切除再生过程中,存在着明显的氧化应激,线粒体生物合成增加,线粒体分裂/融合平衡偏向分裂,并且这些变化呈现具有一定的时间变化规律,这些变化及规律很可能作为将来调控肝再生的重要的潜在干预靶点。     

The Sod Like Activity of Copper: Arnosine,Copper: Anserine and Copper: Homocarnosine Complexes

Carnosine, anserine and homocarnosine are natural compounds which are present in high concentrations (2–20 mM) in skeletal muscles and brain of many vertebrates. We have demonstrated in a previous work that these compounds can act as antioxidants, a result of their ability to scavenge peroxyl radicals, singlet oxygen and hydroxyl radicals. Carnosine and its analogues have been shown to be efficient chelating agents for copper and other transition metals. Since human skeletal muscle contains one-third of the total copper in the body (20–47 mmol/kg) and the concentration of carnosine in this tissue is relatively high, the complex of carnosine:copper may be of biological importance. We have studied the ability of the coppenarnosine (and other carnosine derivatives) complexes to act as superoxide dismutasc. The results indicate that the complex of copper:carnosine can dismute superoxide radicals released by neutrophils treated with PMA in an analogous mechanism to other amino acids and copper complexes. Copper:anserine failed to dismute superoxide radicals and coppwhomocarnosine complex was efficient when the cells were treated with PMA or with histone-opsonized streptococci and cytochalasine B. The possible role of these compounds to act as physiological antioxidants that possess superoxide dismutase activity is discussed.     

Mechanisms of Liver Injury. I. TNF-alpha-induced liver injury: role of IKK, JNK, and ROS pathways

TNF-alpha activates several intracellular pathways to regulate inflammation, cell death, and proliferation. In the liver, TNF-alpha is not only a mediator of hepatotoxicity but also contributes to the restoration of functional liver mass by driving hepatocyte proliferation and liver regeneration. This review summarizes recent advances in TNF-alpha signaling mechanisms that demonstrate how the IKK, ROS, and JNK pathways interact with each other to regulate hepatocyte apoptosis and proliferation. Activation of these pathways is causatively linked to liver injury induced by concanavalin A, TNF-alpha, and ischemia-reperfusion and to liver regeneration and hepatocarcinogenesis. In light of recent findings, pharmacological inhibitors of JNK and IKK and antioxidants may be promising new tools for the treatment of hepatitis, ischemia-reperfusion injury, and hepatocellular carcinoma.     

Effects of Sclerin on Energy-linked Functions and Phospholipase Activity in Mitochondria Isolated from Rat Liver and Some Plants

Sclerin (SCL) not only elevated the respiratory control ratio and ADP/O ratio in mitochondria isolated from rat liver and some plants, but was effective in maintaining the energy-linked functions in these mitochondria during aging. There was a close relationship in the effect of SCL between the liberation of fatty acid and maintenance of the energy-linked functions in mitochondria during aging. The liberation of fatty acid was mainly due to the digestion of mitochondrial phospholipids by endogenous phospholipase. SCL had no effect on the activity of phospholipase and rather raised the level of endogenous phospholipase in mitochondria during aging at 30°C. The activity of phospholipase in mitochondria was inhibited by ATP, but stimulated by DNP. It was supposed that SCL inhibits the activity of phospholipase through ATP or high-energy intermediates which is maintained in mitochondria during aging. SCL had a protective effect on the activity of DNP-activated ATPase in mitochondria stored in the cold, and, at a very low concentration, stimulated the ATP-driven NAD reduction by mitochondria.     

Response of Isolated Rat Liver Mitochondria to Variation of External Osmolarity in KCl Medium: Regulation of Matrix Volume and Oxidative Phosphorylation

When isolated rat liver mitochondria are incubated in KCl medium, matrix volume, flux, and forces in both hypo- and hyperosmolarity are time-dependent. In hypoosmotic KCl medium, matrix volume is regulated via the K⁺/H⁺ exchanger. In hyperosmotic medium, the volume is regulated in such a manner that at steady state, which is reached within 4 min, it is maintained whatever the hyperosmolarity. This regulation is Pi- and -dependent, indicating Pi-K salt entry into the matrix. Under steady state, hyperosmolarity has no effect on isolated rat liver mitochondria energetic parameters such as respiratory rate, proton electrochemical potential difference, and oxidative phosphorylation yield. Hypoosmolarity decreases the NADH/NAD⁺ ratio, state 3 respiratory rate, and , while oxidative phosphorylation yield is not significantly modified. This indicates kinetic control upstream the respiratory chain. This study points out the key role of potassium on the regulation of matrix volume, flux, and forces. Indeed, while matrix volume is regulated in NaCl hyperosmotic medium, flux and force restoration in hyperosmotic medium occurs only in the presence of external potassium.     

Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production,while the Inhibition of Glycolysis Has Less Impact on Sperm Motility

Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37°C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence.     

Effects of Anions on Swelling, Respiration, and Phosphorylation of Isolated Corn Mitochondria

The effects of a series of anions on swelling, respiration, and oxidative phosphorylation of corn mitochondria were studied. Active mitochondrial swelling similar to that found with HPO₄⁻² was demonstrated in the presence of IO₃⁻, NO₂⁻, MoO₄⁻², SO₄⁻², HAsO₄⁻², acetate, S₂O₃⁻², SeO₄⁻², CrO₄⁻², and WoO₄⁻². In general, those anions which caused active swelling also released respiration and reduced the efficiency of oxidative phosphorylation with exogenous NADH as substrate. The degree of passive swelling in the presence of certain of the monovalent anions was found to approximate the order of the lyotropic series (SCN⁻ > CIO₄⁻ > I⁻ > NO₃⁻ > CI⁻).