Ameliorative effect of ursolic acid on ochratoxin A-induced renal cytotoxicity mediated by Lonp1/Aco2/Hsp75.

Ochratoxin A (OTA) is a mycotoxin ubiquitous in feeds and foodstuffs. The water-insoluble pentacyclic triterpene bioactive compound, ursolic acid (UA), is widespread in various cuticular waxes of edible fruits, food materials, and medicinal plants. Although studies have reported that oxidative stress was involved in both the nephrotoxicity of OTA and the renoprotective function of UA, the role of stress-responsive Lon protease 1 (Lonp1) in the renoprotection of UA against OTA is still unknown. In this study, cell viability, reactive oxygen species (ROS) production, and several proteins' expressions of human embryonic kidney 293T (HEK293T) cells in response to UA, OTA, and/or Lonp1 inhibitor CDDO-me treatment were detected to reveal the protective mechanism of UA against OTA-induced renal cytotoxicity. Results indicated that a 2 h-treatment of 1 µM UA could significantly alleviate the ROS production and cell death induced by a 24 h-treatment of 8 µM OTA in HEK293T cells (P < 0.05). Compared with the control, the protein expressions of Lonp1, Aco2 and Hsp75 were significantly inhibited after 8 µM OTA treating for 24 h (P < 0.05), which could be notably reversed by the pre-treatment and post-treatment of 1 µM UA (P < 0.05). The protein expressions of Lonp1, Aco2 and Hsp75 were inhibited by the addition of CDDO-me. The three protein expression trends were similar before and after the addition of CDDO-me. In conclusion, OTA could inhibit the expression of Lonp1, suppressing Aco2 and Hsp75 as a result, thereby activating ROS and inducing cell death in HEK293T cells, which could be alleviated by UA pre-treatment.

The bactericidal effect of an ionizer under low concentration of ozone.

BACKGROUND: Several mechanisms have been suggested for the bactericidal action of ionizers including electrical phenomena, effects of negative and positive ions and electrostatic repulsion. Negative and positive ions have indeed been shown to have bactericidal effects. In addition, since ozone is generated along with ions, these may contribute to the bacterial killing. In this study, we used a newly developed ionizer, which generates a relatively low concentration of ozone, to determine whether its effect on bacterial cells were due to ions or ozone, and, if ions, how the ions exerted their effects. RESULTS: The effect of ions on bacterial killing was compared with that of the ozone produced using an ion trap to remove the ions. The ionizer had the ability to kill the bacteria, and ion capture dramatically reduced its bactericidal effect, indicating that the ozone generated had little or no bactericidal effect under these conditions, and the ions produced were responsible for almost all the bacterial killing. Operation of the ionizer increased the level of 8-oxo-dG, a marker of oxidative DNA damage, and decreased aconitase activity, which is known to be sensitive to ROS. The ionizer further affected the adenylate energy charge of bacterial cells. Removal of the ions with the ion trap greatly reduced all these effects. CONCLUSION: These results indicate that negative and positive ions generated by the ionizer are responsible for inducing oxidative stress and so reducing bacterial survival.

Nitric oxide-dependent killing of aerobic, anaerobic and persistent Burkholderia pseudomallei.

Burkholderia pseudomallei infections are fastidious to treat with conventional antibiotic therapy, often involving a combination of drugs and long-term regimes. Bacterial genetic determinants contribute to the resistance of B. pseudomallei to many classes of antibiotics. In addition, anaerobiosis and hypoxia in abscesses typical of melioidosis select for persistent populations of B. pseudomallei refractory to a broad spectrum of antibacterials. We tested the susceptibility of B. pseudomallei to the drugs hydroxyurea, spermine NONOate and DETA NONOate that release nitric oxide (NO). Our investigations indicate that B. pseudomallei are killed by NO in a concentration and time-dependent fashion. The cytoxicity of this diatomic radical against B. pseudomallei depends on both the culture medium and growth phase of the bacteria. Rapidly growing, but not stationary phase, B. pseudomallei are readily killed upon exposure to the NO donor spermine NONOate. NO also has excellent antimicrobial activity against anaerobic B. pseudomallei. In addition, persistent bacteria highly resistant to most conventional antibiotics are remarkably susceptible to NO. Sublethal concentrations of NO inhibited the enzymatic activity of [4Fe-4S]-cofactored aconitase of aerobic and anaerobic B. pseudomallei. The strong anti-B. pseudomallei activity of NO described herein merits further studies on the application of NO-based antibiotics for the treatment of melioidosis.

Short-term effects of zinc on acetylcholine metabolism and viability of SN56 cholinergic neuroblastoma cells.

Excessive accumulation of zinc in the brain is one of putative factors involved in pathomechanism of cholinergic encephalopathies. The aim of this work was to investigate whether short-term increase of zinc concentration in the extracellular space may affect energy and acetylcholine metabolism in SN56 cholinergic cells of septal origin. Short 30 min exposition of SN56 cells to increasing zinc levels caused greater loss of viability of differentiated (DC, [EC(0.4)] 0.09 mM) than nondifferentiated cells (NC, [EC(0.4)] 0.14 mM). Concentration-dependent accumulation of zinc displayed exponential non-saturable kinetics. Zinc accumulation caused the decrease of calcium accumulation in mitochondria and its increase in cytoplasmic compartment of SN56 cells. Significant inverse and direct correlations were found between zinc accumulation and calcium levels in mitochondrial (r=-0.96, p=0.028) and cytoplasmic (r=0.97, p=0.028) compartments of DC, respectively. Zinc exerted similar inhibition of pyruvate dehydrogenase, aconitase and isocitrate dehydrogenase both in NC and DC homogenates, at Ki values equal to about 0.07, 0.08 and 0.005 mM, respectively. On the other hand, ketoglutarate dehydrogenase activity in DC was inhibited by zinc (Ki 0.0005 mM) 8 times stronger that in NC (Ki 0.004 mM). Also zinc-evoked decreases in acetylcholine content and its release were significantly greater in DC than in NC. Same conditions caused suppression of cytoplasmic and mitochondrial content of acetyl-CoA, that positively correlated with inhibition of transmitter functions (r=0.995, p=005) and loss of cell viability (r=0.990, p=0.0006), respectively. Significant correlations were also found in zinc-challenged cells between pyruvate dehydrogenase activity and both mitochondrial acetyl-CoA content and cell viability. These data indicate that pyruvate dehydrogenase-dependent acetyl-CoA synthesis in neuronal mitochondria may be a primary target for short-term neurotoxic effects of zinc. In consequence, shortages of acetyl-CoA in the mitochondrial compartment would cause fast loss of functional and structural integrity of cholinergic neurons.

Deferiprone targets aconitase: implication for Friedreich's ataxia treatment.

BACKGROUND: Friedreich ataxia is a neurological disease originating from an iron-sulfur cluster enzyme deficiency due to impaired iron handling in the mitochondrion, aconitase being particularly affected. As a mean to counteract disease progression, it has been suggested to chelate free mitochondrial iron. Recent years have witnessed a renewed interest in this strategy because of availability of deferiprone, a chelator preferentially targeting mitochondrial iron. METHOD: Control and Friedreich's ataxia patient cultured skin fibroblasts, frataxin-depleted neuroblastoma-derived cells (SK-N-AS) were studied for their response to iron chelation, with a particular attention paid to iron-sensitive aconitase activity. RESULTS: We found that a direct consequence of chelating mitochondrial free iron in various cell systems is a concentration and time dependent loss of aconitase activity. Impairing aconitase activity was shown to precede decreased cell proliferation. CONCLUSION: We conclude that, if chelating excessive mitochondrial iron may be beneficial at some stage of the disease, great attention should be paid to not fully deplete mitochondrial iron store in order to avoid undesirable consequences.

Involvement of glia in central sensitization in trigeminal subnucleus caudalis (medullary dorsal horn).

Central sensitization is a crucial mechanism underlying the increased excitability of nociceptive pathways following peripheral tissue injury and inflammation. We have previously demonstrated that the small-fiber excitant and inflammatory irritant mustard oil (MO) applied to the tooth pulp produces glutamatergic- and purinergic-dependent central sensitization in brainstem nociceptive neurons of trigeminal subnucleus caudalis (Vc). Recent studies have implicated both astrocytes and microglia in spinal nociceptive mechanisms, showing, for example, that inhibition of spinal astroglial metabolism or spinal microglial p38MAPK activation can attenuate hyperalgesia in inflammatory pain models but have not tested effects of glial inhibitors on central sensitization in functionally identified spinal nociceptive neurons. The aim of the present study was to determine whether glial cells are involved in the MO-induced central sensitization in Vc nociceptive neurons, by examining the effects of intrathecally applied SB203580 (SB), an inhibitor of p38MAPK, and fluoroacetate (FA), an inhibitor of the astroglial metabolic enzyme aconitase. During continuous superfusion of phosphate-buffered saline over Vc, MO application to the pulp-induced central sensitization in Vc nociceptive neurons reflected in significant increases in cutaneous mechanoreceptive field (RF) size and responses to noxious mechanical stimuli and a decrease in mechanical activation threshold. The i.t. application of SB or FA markedly attenuated the MO-induced increases in pinch RF size and responses to noxious stimuli and the decrease in activation threshold. Neither SB nor FA application significantly affected the baseline (i.e., pre-MO application) RF and response properties. These results suggest that glial metabolic processes are important in the development of Vc central sensitization.

[Studies of interaction of intracellular signalling and metabolic pathways under inhibition of mitochondrial aconitase with fluoroacetate].

Mitochondrial aconitase has been shown to be inactivated by a spectrum of substances or critical states. Fluoroacetate (FA) is the most known toxic agent inhibiting aconitase. The biochemistry of toxic action of FA is rather well understood, though no effective therapy has been proposed for the past six decades. In order to reveal novel approaches for possible antidotes to be developed, experiments were performed with rat liver mitochondria, Ehrlich ascite tumor cells and cardiomyocytes, exposed to FA or fluorocitrate in vitro. The effect of FA developed at much higher concentrations in comparison with fluorocitrate and was dependent upon respiratory substrates in experiments with mitochondria: with pyruvate, FA induced a slow oxidation and/or leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides was prevented by incubation of mitochondria with cyclosporin A. Studies of the pyridine nucleotides level and calcium response generated in Ehrlich ascite tumor cells under activation with ATP also revealed a loss of pyridine nucleotides from mitochondria resulting in a shift in the balance of mitochondrial and cytosolic NAD(P)H under exposure to FA. An increase of cytosolic [Ca2+] was observed in the cell lines exposed to FA and is explained by activation of plasma membrane calcium channels; this mechanism, could have an impact on amplitude and rate of Ca2+ waves in cardiomyocytes. Highlighting the reciprocal relationship between intracellular pyridine nucleotides and calcium balance, we discuss metabolic pathway modulation in the context of probable development of an effective therapy for FA poisoning and other inhibitors of aconitase.

Effects of potassium-magnesium citrate supplementation on cytosolic ATP citrate lyase and mitochondrial aconitase activity in leukocytes: a window on renal citrate metabolism.

BACKGROUND: An increase in urinary citrate excretion is associated with a decrease in activity of renal cortical cytosolic ATP citrate lyase (ACL) and mitochondrial aconitase (m-aconitase). Because potassium-magnesium citrate causes an increase in urinary citrate excretion, we decided to assess its effects on ACL and m-aconitase in the leukocytes of renal stone patients. METHODS: Twenty male renal stone patients were supplemented with potassium-magnesium citrate twice daily (i.e. 42 mEq potassium, 21 mEq magnesium, and 63 mEq citrate per day) for a period of 1 month. Two 24-h urine and one 15-mL heparinized blood samples were collected from each patient before and after supplementation. Urine samples were analyzed for relevant biochemical compositions. Leukocytes were separated from blood samples by centrifugation and assayed for ACL and m-aconitase activity. RESULTS: Supplementation with potassium-magnesium citrate significantly increased urinary pH (P < 0.005) and excretions of potassium (P < 0.001), magnesium (P < 0.001) and citrate (P < 0.0001). The activity of both ACL and m-aconitase were significantly decreased (P < 0.004 and P < 0.02 respectively). The decrease in ACL activity was inversely correlated with an increase in urinary excretion of both potassium (r = -0.620, P < 0.0001) and citrate (r = -0.451, P < 0.004). A similar inverse correlation was observed between m-aconitase activity and urinary excretion of citrate (r = -0.322, P < 0.043). CONCLUSION: Changes in enzyme activity, related to citrate metabolism in leukocytes, might reflect the status of renal tubular cells.

1-Methyl-4-phenylpyridinium (MPP+)-induced apoptosis and mitochondrial oxidant generation: role of transferrin-receptor-dependent iron and hydrogen peroxide.

1-Methyl-4-phenylpyridinium (MPP(+)) is a neurotoxin used in cellular models of Parkinson's Disease. Although intracellular iron plays a crucial role in MPP(+)-induced apoptosis, the molecular signalling mechanisms linking iron, reactive oxygen species (ROS) and apoptosis are still unknown. We investigated these aspects using cerebellar granule neurons (CGNs) and human SH-SY5Y neuroblastoma cells. MPP(+) enhanced caspase 3 activity after 24 h with significant increases as early as 12 h after treatment of cells. Pre-treatment of CGNs and neuroblastoma cells with the metalloporphyrin antioxidant enzyme mimic, Fe(III)tetrakis(4-benzoic acid)porphyrin (FeTBAP), completely prevented the MPP(+)-induced caspase 3 activity as did overexpression of glutathione peroxidase (GPx1) and pre-treatment with a lipophilic, cell-permeable iron chelator [N, N '-bis-(2-hydroxybenzyl)ethylenediamine-N, N '-diacetic acid, HBED]. MPP(+) treatment increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling)-positive cells which was completely blocked by pre-treatment with FeTBAP. MPP(+) treatment significantly decreased the aconitase and mitochondrial complex I activities; pre-treatment with FeTBAP, HBED and GPx1 overexpression reversed this effect. MPP(+) treatment increased the intracellular oxidative stress by 2-3-fold, as determined by oxidation of dichlorodihydrofluorescein and dihydroethidium (hydroethidine). These effects were reversed by pre-treatment of cells with FeTBAP and HBED and by GPx1 overexpression. MPP(+)-treatment enhanced the cell-surface transferrin receptor (TfR) expression, suggesting a role for TfR-induced iron uptake in MPP(+) toxicity. Treatment of cells with anti-TfR antibody (IgA class) inhibited MPP(+)-induced caspase activation. Inhibition of nitric oxide synthase activity did not affect caspase 3 activity, apoptotic cell death or ROS generation by MPP(+). Overall, these results suggest that MPP(+)-induced cell death in CGNs and neuroblastoma cells proceeds via apoptosis and involves mitochondrial release of ROS and TfR-dependent iron.

Complexities in the neurotoxic actions of 6-hydroxydopamine in relation to the cytoprotective properties of taurine.

The neurotoxin 6-hydroxydopamine was shown to cause an imbalance between the direct and indirect pathways of the striato-nigral system as evidenced by a decreased release of gamma-aminobutyric acid and taurine in the substantia nigra but not in the globus pallidus following neostriatal stimulation with kainate (100 microM). The neurotoxicity of 6-hydroxydopamine is generally believed to result from reactive-oxygen radical formation, although it is also known to inhibit mitochondrial NADH dehydrogenase. The release of Fe(II) from the unactivated form [3Fe(III)-4S] of cytoplasmic aconitase (EC(50) < 8 microM) was shown to be followed by the slower oxidation of thiol groups in the protein. Complete loss of -SH groups, and enzyme activity, was seen after incubation of glyceraldenyde-3-phosphate dehydrogenase with 200 microM 6-hydroxydopamine for 75 min at 37 degrees C (IC(50) = 70.8 +/- 0.3 microM). Thus the cellular effects of 6-hydroxydopamine are complex, involving impairment of mitochondrial function, iron- release, sulphydryl-group oxidation, and enzyme inhibition in addition to direct generation of reactive oxygen radicals. Taurine, which is known to be neuroprotective in some other systems, only affords protection against some of these effects, thereby explaining its reported ineffectiveness against 6-hydroxydopamine toxicity.

Aconitase activity in rat liver.

This paper presents data about the subcellular distribution of aconitases in rat liver and some properties of the aconitase activity in cytosol, mitochondria and soluble mitochondrial protein (SMP). The cytosolic and mitochondrial aconitase activity was 64.8% or 61.0% and 20.1% or 19.4% of the total rat liver aconitase activity when cis-aconitate or isocitrate was used as substrate. Aconitase activity of stored SMP and mitochondria with phosphate buffer (pH 7.4) and 0.25 M sucrose (pH 7.4) as isolation medium respectively, was reduced to an equal extent upon exposure to air. Fresh SMP preparations immediately and three hr after isolation had the same aconitase activity. It is concluded that phosphate has no role in the oxidative degradation of mitochondrial aconitase and does not inhibit it. Complete restoration of the decreased mitochondrial aconitase activity to the initial level was achieved with thiomalate and Fe2+ under anaerobic conditions or 60-70% was restored during the long period (60 min) of incubation with exogenous substrate. The aconitase activity of cytosol and mitochondria increased upon exposure to air for 7 1/2 hr. This finding is interpreted to suggest the existence of putative aconitase activity.