Live-Cell Assessment of Reactive Oxygen Species Levels Using Dihydroethidine.

Reactive oxygen species (ROS) play an important role in cellular (patho)physiology. Empirical evidence suggests that mitochondria are an important source of ROS, especially under pathological conditions. Here, we describe a method for ROS measurement using dihydroethidium (HEt) and live-cell microscopy.

Multiorgan Development of Oxidative and Nitrosative Stress in LPS-Induced Endotoxemia in C57Bl/6 Mice: DHE-Based In Vivo Approach.

Detection of free radicals in tissues is challenging. Most approaches rely on incubating excised sections or homogenates with reagents, typically at supraphysiologic oxygen tensions, to finally detect surrogate, nonspecific end products. In the present work, we explored the potential of using intravenously (i.v.) injected dihydroethidine (DHE) to detect superoxide radical (O2 ∙-) abundance in vivo by quantification of the superoxide-specific DHE oxidation product, 2-hydroxyethidium (2-OH-E+), as well as ethidium (E+) and DHE in multiple tissues in a murine model of endotoxemia induced by lipopolysaccharide (LPS). LPS was injected intraperitoneally (i.p.), while DHE was delivered via the tail vein one hour before sacrifice. Tissues (kidney, lung, liver, and brain) were harvested and subjected to HPLC/fluorescent analysis of DHE and its monomeric oxidation products. In parallel, electron spin resonance (EPR) spin trapping was used to measure nitric oxide (∙NO) production in the aorta, lung, and liver isolated from the same mice. Endotoxemic inflammation was validated by analysis of plasma biomarkers. The concentration of 2-OH-E+ varied in the liver, lung, and kidney; however, the ratios of 2-OH-E+/E+ and 2-OH-E+/DHE were increased in the liver and kidney but not in the lung or the brain. An LPS-induced robust level of ∙NO burst was observed in the liver, whereas the lung demonstrated a moderate yet progressive increase in the rate of ∙NO production. Interestingly, endothelial dysfunction was observed in the aorta, as evidenced by decreased ∙NO production 6 hours post-LPS injection that coincided with the inflammatory burden of endotoxemia (e.g. elevated serum amyloid A and prostaglandin E2). Combined, these data demonstrate that systemic delivery of DHE affords the capacity to specifically detect O2 ∙- production in vivo. Furthermore, the ratio of 2-OH-E+/E+ oxidation products in tissues provides a tool for comparative insight into the oxidative environments in various organs. Based on our findings, we demonstrate that the endotoxemic liver is susceptible to both O2 ∙--mediated and nonspecific oxidant stress as well as nitrosative stress. Oxidant stress in the lung was detected to a lesser extent, thus underscoring a differential response of liver and lung to endotoxemic injury induced by intraperitoneal LPS injection.

Sterol transfer, PI4P consumption, and control of membrane lipid order by endogenous OSBP.

The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol-binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi network (TGN). Using the inhibitor OSW-1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4-kinase PI4KIIIß triggers large periodic traveling waves of PI4P across the TGN These waves are cadenced by long-range PI4P production by PI4KIIα and PI4P consumption by OSBP Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4-kinases to control the lipid composition of subcellular membranes.

4-Isoxazolyl-1,4-dihydropyridines exhibit binding at the multidrug-resistance transporter.

The 4-isoxazolyl-dihydropyridines (IDHPs) exhibit inhibition of the multidrug-resistance transporter (MDR-1), and exhibit an SAR distinct from their activity at voltage gated calcium channels (VGCC). Among the four most active IDHPs, three were branched at C-5 of the isoxazole, including the most active analog, 1k.

Hemoperfusion treatment in a septic shock patient with autosomal dominant polycystic kidney disease and increased HMGB1 protein levels.

This case report describes polymyxin B-immobilized fiber (PMX-F) treatment of septic shock caused by pyelonephritis in a 68-year-old woman with autosomal dominant polycystic kidney disease. She was admitted for severe lower left abdominal pain, high fever (40°C) and gross hematuria. Her endotoxin and high-mobility group box-1 protein (HMGB1) levels were extremely elevated. Her blood pressure was 68/36 mm Hg. Urinalysis revealed innumerable white blood cells (WBCs). Blood and urine cultures were positive for Klebsiella pneumoniae and Pseudomonas aeruginosa. Plain abdominal radiography showed large kidney shadows and calcium deposition. Septic shock with endotoxemia was diagnosed. Her symptoms of septic shock persisted for 3 days with antibiotics, γ-globulin and dopamine. Direct hemoperfusion was performed twice with a PMX-F column. The patient's body temperature, WBC count and C-reactive protein level decreased. Her blood endotoxin level and blood HMGB1 level also decreased to an almost normal level. She was discharged on day 23 after admission.

Evaluation of different perfusion durations in direct hemoperfusion with polymyxin B-immobilized fiber column therapy for acute exacerbation of interstitial pneumonias.

BACKGROUND: Recently, the potential therapeutic effect of direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX-DHP) has been reported for acute exacerbation of interstitial pneumonia (AE-IP), a highly morbid clinical event; however, there is no consensus on the appropriate procedure for PMX-DHP. We examined the appropriate perfusion duration of PMX-DHP for AE-IP. METHODS: AE-IP patients receiving PMX-DHP were divided into two groups: short-duration group (≤6 h) (n = 5) and long-duration group (12 h) (n = 12). RESULTS: ThePaO(2)/FiO(2) (P/F) ratio increased immediately after PMX-DHP in the two groups. In the long-duration group, the P/F ratio continued to increase over the following 7 days, while, in the short-duration group, the P/F ratio declined again 3 days after therapy. The survival rate 30 days after PMX-DHP was significantly higher in the long-duration group than in the short-duration group. CONCLUSIONS: A long perfusion duration of PMX-DHP is more efficacious for AE-IP than a short perfusion duration.

Superoxide radical formation in diverse organs of rats with experimentally induced obstructive jaundice.

Oxidative stress seems to be a cardinal feature of cholestasis, implicated in the pathophysiology of organ injury not only in the liver, but also in several extrahepatic tissues. The present study was designed to assess directly oxidative stress in vital organs of experimentally jaundiced rats by measuring the key oxidative stress marker superoxide radical (O2(*-)). Twelve male Wistar rats underwent laparotomy and were divided into two groups - group I (n = 6) sham operated, and group II (n = 6) bile-duct ligated. Ten days later, the O2(*-) formation rate was quantified in liver, intestine, kidney and heart of all animals. These measurements were done by application of a new ultrasensitive fluorescent assay for the in vivo quantification of O2(*-), which is based on the 1:1 molar stoichiometric reaction of O2(*-) with dihydroethidine (DHE, an O2(*-) trap) that results in the formation of the specific product 2-OH-ethidium. 2-OH-Ethidium was measured by fluorescence in rats' organs and its formation rate was converted to O2(*-) production rate. As compared to sham-operated rats, in jaundiced rats there was a significant increase of O2(*-) in the intestine (136%, P < 0.01), liver (104%, P < 0.01), and kidney (95%, P < 0.01), whereas there was no significant difference in heart O2(*-) levels. Superoxide radical may play an important role in the pathophysiology of cholestatic liver injury, intestinal barrier failure and renal failure, associated with postoperative morbidity and mortality in obstructive jaundice. On the contrary, O2(*-) and oxidative stress are possibly not implicated in the pathophysiology of hepatic cardiomyopathy.

[Antioxidant activity of inhibitors of free radical reactions in the dressing materials for wound healing].

Antioxidant activity (AA) of inhibitors of free radical reactions (FRR) (dieton, mexidol, trypsin), aplied to the dressing material for wound healing was studied. In our work we used a model system containing suspension of laminated liposome, formed from fraction of total chicken yolk phospholipids. Lipid peroxidation (LPO) of liposome membranes was initiated by addition of Fe2+ ions. The kinetics of FRR was followed by coumarine-enhanced chemiluminescence (CL). It was found that AA of the inhibitors was determined by their ability to intersept aqueous and hydrofobic free radicals and chelate Fe2+ ions. Their ability to intersept radicals reduced in the following order: dieton > trypsin > mexidol. In addition we discovered unknown ability of mexidol to interact with Fe2+, that resulted in elemination of FRR catalyst. Investigating AA of the FRR inhibitors in the two-components mixture, consisting of dieton and mexidol, we observed the effect of multifunctionality: dieton, increased the duration of latent period of CL by intersepting lipid peroxyl radicals, while mexidol, decreased its value by interacting with Fe2+, i.e. mexidol masked the action of dieton. Investigating AA of two-components mixture, consisting of mexidol and trypsine, we observed the same effect of multifunctionality. In the two-component mixture, consisting of trypsine and dieton, the action of the inhibitors was found to be synergistic. All antioxidant properties of these FRR inhibitors were also preserved in the three component mixture. Hence, mixture components, dieton, mexidol and trypsin, possess high AA, that validates their use in dressing materials employed for wound healing.

Inhibitory effect of pranidipine on N-type voltage-dependent Ca2+ channels in mice.

We investigated the N-type voltage-dependent calcium channel blocking action of pranidipine, a novel dihydropyridine (DHP) derivative. Pranidipine significantly suppressed KCl-induced intracellular calcium changes ([Ca(2+)](i)) in a dose-dependent fashion in dorsal root ganglion neurons. A patch-clamp investigation revealed a dose-dependent blocking effect on N-type currents. Intrathecal injection of pranidipine significantly shortened the licking time in the late phase of the formalin test, as occurs with cilnidipine and amlodipine, which act on L- and N-type channels. Conversely, nicardipine, which acts exclusively on L-type channels, had no antinociceptive effect. Our results indicate that pranidipine inhibits N-type calcium channels. Furthermore, it exerts an antinociceptive effect, which might be related to an attenuation of synaptic transmission by nociceptive neurons due to the blocking effect of pranidipine on N-type calcium channels in primary nociceptive afferent fibers.

Bile acid-induced Mallory body formation in drug-primed mouse liver.

Chronic cholestasis is associated with retention of bile acids and profound cytoskeletal alterations in hepatocytes including Mallory body (MB) formation. The mechanisms responsible for MB formation in cholestatic liver diseases are unclear. The aim of our study was to determine the relevance of cholestasis and bile acids for MB formation. For this purpose mice received a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-supplemented diet for 2.5 months to induce MB formation. After recovery from DDC intoxication for 4 weeks followed by disappearance of MBs, these drug-primed mice were subjected to DDC refeeding, common bile duct ligation (CBDL), and feeding of a cholic acid (CA)-supplemented diet for 7 days, respectively. Cytokeratin (CK) 8 and CK 18 expression was studied by competitive reverse transcriptase-polymerase chain reaction and Western blot analysis. Cytoskeletal alterations of hepatocytes and MB formation were monitored by immunofluorescence microscopy and immunohistochemistry using CK-, ubiquitin-, and MB-specific antibodies. Like DDC refeeding, both CBDL and CA feeding of drug-primed mice significantly increased CK 8 and CK 18 mRNA and protein levels (with excess of CK 8) and resulted in ubiquitination and abnormal phosphorylation of CKs. Furthermore, CBDL and CA feeding resulted in rapid neoformation of MBs in drug-primed mice. It is concluded that MB formation in cholestatic liver diseases may be triggered by the action of potentially toxic bile acids.

pH-dependent one- and two-electron oxidation of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine catalyzed by horseradish peroxidase.

The porphyrinogenic agent 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) is known to inactivate hepatic cytochrome P450 (P450) enzymes 2C11, 2C6, and 3A1 [Correia et al. (1987) Arch. Biochem. Biophys. 258, 436-451] by different mechanisms. The inactivation of P450 2C11 and 2C6 appears to be due to the ethylation of the heme in the active sites of the enzymes [Augusto et al. (1982) J. Biol. Chem. 257, 11288-11295], whereas the inactivation of P450 3A1 appears to involve the covalent binding of the heme to the apoprotein [Correia et al. (1987)]. Moreover, we have found that DDEP inactivates horseradish peroxidase (HRP) pretreated with hydrogen peroxide. In this system, DDEP was oxidized predominately to 3,5-dicarbethoxy-2,6-dimethyl-4-ethylpyridine (EDP) under weakly acidic conditions and predominately to 3,5-dicarbethoxy-2,6-dimethylpyridine (DP) under basic conditions. The loss of heme and the formation of altered heme products were also pH-dependent and were correlated with the formation of DP and the inactivation of HRP. Thus the inactivation of HRP appears to depend on the formation of an ethyl radical, which presumably reacts with the heme in the active site of the enzyme. Similar product ratios were obtained for the oxidation of DDEP by K3Fe(CN)6, indicating that product ratios of DP over EDP are mainly determined by the pH of buffer. These results, in addition to semiemperical calculations (AM1) for the oxidation of DDEP in the gas phase, are consistent with the idea that the inhibitor undergoes a single-electron oxidation to form the DDEP radical cation, the fate of which depends on the environment of the active site of the enzyme. The proposed formation of a radical cation by the abstraction of an electron from nitrogen is consistent with the finding of low intramolecular isotope effects of the metabolism of 3,5-dicarbethoxy-2,6-dimethyl-[4-2H,4-1H]-1,4-dihydropyridine by P450 2C11 and 3A4. Under basic or aprotic conditions, the radical dissociates to form DP and the ethyl radical, which reacts with the heme, thereby inactivating the enzyme. Under acidic or polar conditions, the radical undergoes an additional one-electron oxidation to form EDP.(ABSTRACT TRUNCATED AT 400 WORDS)