Neuroprotective Properties of Quinone Reductase 2 Inhibitor M-11, a 2-Mercaptobenzimidazole Derivative.

The ability of NQO2 to increase the production of free radicals under enhanced generation of quinone derivatives of catecholamines is considered to be a component of neurodegenerative disease pathogenesis. The present study aimed to investigate the neuroprotective mechanisms of original NQO2 inhibitor M-11 (2-[2-(3-oxomorpholin-4-il)-ethylthio]-5-ethoxybenzimidazole hydrochloride) in a cellular damage model using NQO2 endogenous substrate adrenochrome (125 µM) and co-substrate BNAH (100 µM). The effects of M-11 (10-100 µM) on the reactive oxygen species (ROS) generation, apoptosis and lesion of nuclear DNA were evaluated using flow cytometry and single-cell gel electrophoresis assay (comet assay). Results were compared with S29434, the reference inhibitor of NQO2. It was found that treatment of HT-22 cells with M-11 results in a decline of ROS production triggered by incubation of cells with NQO2 substrate and co-substrate. Pre-incubation of HT-22 cells with compounds M-11 or S29434 results in a decrease of DNA damage and late apoptotic cell percentage reduction. The obtained results provide a rationale for further development of the M-11 compound as a potential neuroprotective agent.

Multiple targeted drugs carrying biodegradable membrane barrier: anti-adhesion, hemostasis, and anti-infection.

A multiple targeted drug carrying bilayer membrane for preventing an abdominal adhesion is prepared by electrospinning. Two bioactive drugs were successfully incorporated into this bilayer membrane and can be independently released from nanofibrous scaffolds without losing structural integrity and functionality of the anti-adhesion membrane. Besides, the drug release profile could be easily adjusted by optimizing the swelling behavior of the fibrous scaffold. The inner layer of the bilayered fibrous membranes loaded with carbazochrome sodium sulfonate (CA) showed an excellent vascular hemostatic efficacy and formed little clot during in vivo experiment. The outer layer loaded with tinidazole (TI) had outstanding antibacterial effect against the anaerobe. We believe this approach could serve as a model technique to guide the design of implants with drug delivery functions.

Free radical signalling underlies inhibition of CaV3.2 T-type calcium channels by nitrous oxide in the pain pathway.

Nitrous oxide (N2O, laughing gas) has been used as an anaesthetic and analgesic for almost two centuries, but its cellular targets remain unclear. Here, we present a molecular mechanism of nitrous oxide's selective inhibition of CaV3.2 low-voltage-activated (T-type) calcium channels in pain pathways. Using site-directed mutagenesis and metal chelators such as diethylenetriamine pentaacetic acid and deferoxamine, we reveal that a unique histidine at position 191 of CaV3.2 participates in a critical metal binding site, which may in turn interact with N2O to produce reactive oxygen species (ROS). These free radicals are then likely to oxidize H191 of CaV3.2 in a localized metal-catalysed oxidation reaction. Evidence of hydrogen peroxide and free radical intermediates is given in that N2O inhibition of CaV3.2 channels is attenuated when H2O2 is neutralized by catalase. We also use the adrenochrome test as an indicator of ROS in vitro in the presence of N2O and iron. Ensuing in vivo studies indicate that mice lacking CaV3.2 channels display decreased analgesia to N2O in response to formalin-induced inflammatory pain. Furthermore, a superoxide dismutase and catalase mimetic, EUK-134, diminished pain responses to formalin in wild-type mice, but EUK-134 and N2O analgesia were not additive. This suggests that reduced ROS levels led to decreased inflammation, but without the presence of ROS, N2O was not able to provide additional analgesia. These findings reveal a novel mechanism of interaction between N2O and ion channels, furthering our understanding of this widely used analgesic in pain processing.

Catecholamine oxidation-mediated transcriptional inhibition in Mn neurotoxicity.

Manganese (Mn) poisoning may result in a neurological disorder called manganism. Although the neurotoxic mechanism of Mn is unclear, oxidative stress may be involved based on the interactions between neurotransmitter catecholamines and metals such as iron. Here, we propose a novel mechanism in which Mn oxidizes catecholamines and inhibits cellular transcription. Mn accelerated the oxidation of adrenaline (Ad) and produced adrenochrome (AdC) more effectively than iron. Furthermore, the oxidation of DNA bases increased when Ad, Mn, and iron were present. However, despite the absence of iron, cell viability decreased in the presence of AdC or Ad with Mn, which suggests there is another mechanism independent of oxidative DNA damage. AdC or preincubated Ad with Mn reduced mRNA synthesis in T7 RNA polymerase-driven transcription. RNA synthesis decreased in AdC-treated cells dose-dependently. These results show that Mn disrupts neuronal function via catecholamine oxidation-mediated transcriptional inhibition.

Rapid methods for high-throughput detection of sulfoxides.

Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.

Mechanism and impact of catecholamine conversion by Vibrio cholerae.

Bacterial pathogens are influenced by signaling molecules including the catecholamines adrenaline and noradrenaline which are host-derived hormones and neurotransmitters. Adrenaline and noradrenaline modulate growth, motility and virulence of bacteria. We show that adrenaline is converted by the pathogen Vibrio cholerae to adrenochrome in the course of respiration, and demonstrate that superoxide produced by the respiratory, Na+ - translocating NADH:quinone oxidoreductase (NQR) acts as electron acceptor in the oxidative conversion of adrenaline to adrenochrome. Adrenochrome stimulates growth of V. cholerae, and triggers specific responses in V. cholerae and in immune cells. We performed a quantitative proteome analysis of V. cholerae grown in minimal medium with glucose as carbon source without catecholamines, or with adrenaline, noradrenaline or adrenochrome. Significant regulation of proteins participating in iron transport and iron homeostasis, in energy metabolism, and in signaling was observed upon exposure to adrenaline, noradrenaline or adrenochrome. On the host side, adrenochrome inhibited lipopolysaccharide-triggered formation of TNF-α by THP-1 monocytes, though to a lesser extent than adrenaline. It is proposed that adrenochrome produced from adrenaline by respiring V. cholerae functions as effector molecule in pathogen-host interaction.

The epinephrine assay for superoxide: why dopamine does not work.

Superoxide oxidizes epinephrine to a semiquinone, initiating a series of reactions leading to the colored product adrenochrome. This popular assay for superoxide is more sensitive at higher pH, and it does not work if dopamine is used instead of epinephrine. A kinetic analysis shows that these effects can be explained by competing reactions that lower the yield of the observed product. The catecholamine quinone may cyclize to form the absorbing product, or it may be reduced back to the semiquinone by superoxide. For epinephrine, the quinone cyclizes quickly and adrenochrome formation dominates, but for dopamine, the quinone cyclizes slowly and the back reaction prevails. The yield of adrenochrome increases if the epinephrine semiquinone reacts with O(2) to form more superoxide, but this reaction competes with disproportionation of the semiquinone. Because disproportionation slows as pH increases, both superoxide formation and the yield of adrenochrome increase at higher pH.

Oxidative stress and neurodegeneration: The possible contribution of quinone reductase 2.

There is increasing evidence that oxidative stress is involved in the etiology and pathogenesis of neurodegenerative disorders. Overproduction of reactive oxygen species (ROS) is due in part to the reactivity of catecholamines, such as dopamine, adrenaline, and noradrenaline. These molecules are rapidly converted, chemically or enzymatically, into catechol-quinone and then into highly deleterious semiquinone radicals after 1-electron reduction in cells. Notably, the overexpression of dihydronicotinamide riboside:quinone oxidoreductase (QR2) in Chinese hamster ovary (CHO) cells increases the production of ROS, mainly superoxide radicals, when it is exposed to exogenous catechol-quinones (e.g. dopachrome, aminochrome, and adrenochrome). Here we used electron paramagnetic resonance analysis to demonstrate that the phenomenon observed in CHO cells is also seen in human leukemic cells (K562 cells) that naturally express QR2. Moreover, by manipulating the level of QR2 in neuronal cells, including immortalized neuroblast cells and ex vivo neurons isolated from QR2 knockout animals, we showed that there is a direct relationship between QR2-mediated quinone reduction and ROS overproduction. Supporting this result, the withdraw of the QR2 co-factor (BNAH) or the addition of the specific QR2 inhibitor S29434 suppressed oxidative stress. Taken together, these data suggest that the overexpression of QR2 in brain cells in the presence of catechol quinones might lead to ROS-induced cell death via the rapid conversion of superoxide radicals into hydrogen peroxide and then into highly reactive hydroxyl radicals. Thus, QR2 may be implicated in the early stages of neurodegenerative disorders.

Superoxide dismutase activity in leukocytes.

Superoxide dismutase activity has been identified in both human neutrophils and rabbit alveolar macrophages by two distinct assay procedures. The enzyme is insensitive to both cyanide and azide and is present in the cytosol of the cell. The identification of this enzyme in phagocytic cells is compatible with the theory that superoxide anion might be involved in the bactericidal activity of the cell. It is proposed that the enzyme functions to protect the cell against superoxide generated during the phagocytic process.

Direct and respiratory chain-mediated redox cycling of adrenochrome.

Adrenochrome is reduced by ascorbate in a reaction accompanied by a large and rapid oxygen uptake. The rates of adrenochrome reduction and the concomitant oxygen uptake are decreased in the presence of superoxide dismutase or catalase. The species formed on the one-electron reduction of adrenochrome (i.e., the semiquinone) was shown by pulse radiolysis to rapidly react with oxygen (9.10(8) M-1.s-1), indicating the occurrence of a redox cycling in a system formed by adrenochrome, a reducing agent, and oxygen. Adrenochrome is also reduced to the corresponding semiquinone by complex I of beef heart submitochondrial particles supplemented with NADH, while succinate is unable to support this reduction. The o-semiquinone is the intermediate species in the superoxide-generating cycle resulting from both non-enzymatic and enzymatic reduction. The toxic effects of adrenochrome and its pathophysiological role can be explained, at least in part, on the basis of the demonstrated cycle.

Involvement of carbonyl reductase in superoxide formation through redox cycling of adrenochrome and 9,10-phenanthrenequinone in pig heart.

The effects of adrenochrome, a metabolite of epinephrine (adrenaline), and 9,10-phenanthrenequinone (PQ), a component of diesel exhaust particles, on the stereoselective reduction of 4-benzoylpyridine (4-BP) were examined in pig heart cytosol. PQ was a potent inhibitor for the 4-BP reduction, while adrenochrome was a poor inhibitor. A similar result was observed in the effects of adrenochrome and PQ on the reduction of all-trans retinal. Furthermore, although PQ mediated efficiently the formation of superoxide anion radical through its redox cycling in pig heart cytosol, adrenochrome had no ability to mediate the superoxide formation. These may be because the reactivity for adrenochrome, catalyzed by pig heart carbonyl reductase (PHCR), is much lower than that for PQ. The optimal pH for the reduction of PQ in pig heart cytosol was around 5.5. Dicumarol, a potent inhibitor of DT-diaphorase, had little effect on the time course of NADPH oxidation during the reduction of PQ. Therefore, it is concluded that PHCR plays a critical role in superoxide formation through redox cycling of PQ.

Toxicity of aminochromes.

The first part of the present review deals with the chemical and enzymatic synthesis of adrenochrome and other aminochromes from the corresponding catecholamines. A description of the most significant pathways of formation and the reactivity of the aminochromes is presented. In the second part of the toxicity of aminochromes, mainly at the cardiac and CNS level, is described and some of the molecular mechanisms of the toxic action are outlined. The toxicity of the aminochromes appears to depend mainly on the production of reduced oxygen species through redox cycling. The interaction of aminochromes with sulfhydryl groups and the induced depletion of oxygen, ascorbate and glutathione are additional mechanisms resulting in noxious effects at a cellular level.

Dose-dependent study of liver lipid peroxidation related parameters in rats treated with pp'-DDT.

Rats treated with increasing doses of pp'-DDT (60, 100 and 180 mg/kg body wt.) i.p., for 24 h, showed a dose-independent increase in liver cytochrome P450 levels, together with an increase in lipid peroxidation, measured as production of thiobarbituric acid reactants. This oxidant condition elicited in the liver by DDT was not accompanied by any change in the activity of NADPH-cytochrome c reductase or in the rate of superoxide anion generation by liver microsomal fraction. The activities of superoxide dismutase and glutathione peroxidase were found to be increased in the higher dose DDT-treated rats, without any change in those from catalase and glutathione reductase. The results presented showed an oxidant condition in the liver elicited by DDT treatment of rats, without any adequate hypothesis proposed to explain these data.

Adrenochrome uptake and subcellular distribution in the isolated perfused rat heart.

Adrenochrome uptake and its subcellular distribution were examined using isolated perfused rat heart preparation. The heart was perfused for 30 min with a medium containing 1 to 50 mg/l of 14C-adrenochrome and the subcellular fractions were isolated to measure their radioactivities. A decline in contractile force, a rise in resting tension and an increase in adrenochrome uptake by the heart were seen to depend upon the time of perfusion and the concentration of adrenochrome in the medium. The sarcolemmal fraction had the highest uptake of adrenochrome and this was followed by the microsomal fraction; some accumulation of adrenochrome was also observed in the myofibrillar and mitochondrial fractions. Either 10 or 20 min reperfusion of the heart previously exposed to 25 mg/l of adrenochrome, resulted in approximately 50 or 37% of the radioactivity remaining in the heart; this indicates irreversible binding of adrenochrome to the tissue. Reperfusion of the heart showed restoration of the resting tension but the contractile force did not show any recovery. Propranolol and iproniazid, which have been shown to inhibit the adrenochrome induced cardiotoxicity, reduced adrenochrome uptake by the heart, and prevented adrenochrome-induced depression in contractile force and rise in resting tension. These results indicate that adrenochrome is taken up by the heart and induces cardiac disturbances through its action on different subcellular organelles in the myocardium.

Effect of adrenochrome on adenine nucleotides and mitochondrial oxidative phosphorylation in rat heart.

Effects of adrenochrome, an oxidation product of epinephrine, on myocardial energy production were investigated by studying changes in adenine nucleotide content and mitochondrial oxidative phosphorylation activities in the isolated rat heart. Perfusion of the heart with 50 mg/L adrenochrome induced a marked decline in contractile force within 5 min and this was associated with a rapid decline in the myocardial ATP/AMP ratio. A significant decrease in ATP and ATP/ADP ratio as well as a significant increase in ADP and AMP content was observed at 10 min of perfusion with adrenochrome. Furthermore, mitochondrial oxidative phosphorylation activities were unchanged except that an increase in state 4 respiration and a decrease in RCI value were seen in the heart perfused with adrenochrome for 10 min. Autoradiography of the sections from hearts perfused with 14C-adrenochrome revealed the localization of a significant amount of radioactivity on mitochondria. Adrenochrome at concentrations of 20 mg/L or higher was found to inhibit the oxidative phosphorylation activities of heart mitochondria under in vitro conditions. The depressant effects of adrenochrome on mitochondrial oxidative phosphorylation were additive to those seen with calcium. These data suggest that adrenochrome in the presence of excess calcium in the myocardial cell may impair the process of energy production in mitochondria and this may result in contractile failure of hearts exposed to this cardiotoxic metabolite of epinephrine.

Plasma oxidase assay for screening of myocardial infarction.

The availability of techniques such as surgical reperfusion, angioplasty, and thrombolysis for the treatment of acute myocardial infarction (AMI) has revived interest in seeking an early detectable biochemical marker diagnostic for AMI. Therefore, we investigated whether an unidentified oxidase that is released by activated neutrophils at the onset of AMI could be used as an early diagnostic assay. The conversion by plasma oxidase of 1 microM of adrenaline to 1 microM of adrenochrome represents the plasma oxidase activity (POA) of 1 U/L. Fifty patients suspected of having AMI, 40% of whose electrocardiograms were nondiagnostic for AMI, were admitted to the coronary care unit, and venous blood samples were obtained for determination of the POA and creatine phosphokinase-MB levels. Healthy volunteers (n = 12) served as control subjects, and 8 patients with pneumonia whose leukocyte counts were greater than 15,000 microL were included in the study. In those with AMI (n = 22), as determined by serial creatine phosphokinase-MB, the mean POA (+/- standard error of the mean) was 233 +/- 13 U/L, and in those with angina and no AMI (n = 28) was 127 +/- 5 U/L (P < 0.0001). In the control group, mean POA (+/- standard error of the mean) was 84 +/- 5 U/L (control versus angina; P < 0.01) and for those with infection was 214 +/- 10 U/L. At admission, the creatine phosphokinase-MB was diagnostic for only 12 of the 22 patients with AMI (sensitivity rate of 54%), whereas in 21 of those patients, the POA values were diagnostic for AMI (sensitivity rate of 95%).(ABSTRACT TRUNCATED AT 250 WORDS)

Schizophrenia and cancer: the adrenochrome balanced morphism.

Cancer might be expected to be more common amongst schizophrenics than the general population. They frequently live in selenium deficient regions, have seriously compromised antioxidant defense systems and chain-smoke. The available literature on the cancer-schizoprenia relationship in patients from England, Wales, Ireland, Denmark, USA and Japan, however, strongly suggests that the reverse is true. One of the authors (Hoffer) has treated 4000 schizophrenics since 1952. Only four of these patients has developed cancer. Since low cancer incidence has been recorded amongst patients treated by both conventional physicians using pharmaceuticals and by orthomolecular doctors who emphasize vitamins and minerals, it follows that this depressed cancer incidence must be related to the biochemistry of the disorder itself. Taken as a whole, therefore, the evidence seems to suggest that schizophrenics, their siblings and parents are less susceptible to cancer than the general population. These relationships seem compatible with one or more genetic risk factors for schizophrenia that offer(s) a selective advantage against cancer. There is experimental evidence that appears to support this possibility. Matrix Pharmaceuticals Inc. has received a US patent covering the composition of IntraDose Injectable Gel. This gel contains cisplatin and epinephrine (adrenaline) and is designed to be injected directly into tumour masses. Cisplatin is a very powerful oxidant which will almost certainly rapidly convert the adrenaline to adrenochrome. While the manufacturers of IntraDose consider cisplatin to be the active cytotoxic agent in IntraDose, it seems more likely that adrenochrome and its derivatives may, in fact, be more effective. IntraDose gel has undergone or is undergoing a series of Phase III open-label clinical studies, being injected into patients' tumours that have been identified as the most troublesome by their physicians. The results have been impressive for breast cancer, malignant melanoma, esophageal cancer and cancer of the head, neck and liver. The evidence suggests that there are balanced morphisms in schizophrenia that result in above normal exposure to catecholamine derivatives. Since such catecholamines are both hallucinogenic and anticarcinogenic abnormally high exposure to them simultaneously increases susceptibility to schizophrenia and reduces the probability of developing cancer. These observations have significant implications for the treatment of both illnesses.

Oxidation of 1,1-diphenylhydrazine to N-nitrosodiphenylamine by superoxide radical in the eye.

By microsomes obtained from bovine ciliary body, 1,1-diphenylhydrazine was oxidized to N-nitrosodiphenylamine in the presence of NADPH. This reaction was stimulated by riboflavin which was recognized to be an electron carrier. The oxidizing activity by microsomes was markedly inhibited by superoxide dismutase, but not by SKF 525-A or carbon monoxide. Similarly, the oxidation of 1,1-diphenylhydrazine to its corresponding nitrosamine occurred in varying degrees when the hydrazine derivative was exposed to visible light in the presence of photosensitizers such as riboflavin, flavin adenine dinucleotide, flavin mononucleotide, lumiflavin, lumichrome, NAD+, NADH, NADP+, or NADPH. The photochemical oxidation was inhibited by active oxygen-scavengers such as superoxide dismutase, L-ascorbic acid or alpha-tocopherol. The superoxide radical involved in the photochemical reaction was determined by measuring the oxidation of epinephrine to adrenochrome. The oxidation of epinephrine was well correlated to that of 1,1-diphenylhydrazine. Thus, the present study provided evidence that the superoxide radical is responsible for the oxidation of a hydrazine derivative to a corresponding nitrosamine by ocular tissue microsomes and by photosensitizers.

The role of aminochromes in ultraweak luminescence accompanying oxidative metabolism of catecholamines in model systems in vitro.

Ultraweak luminescence (UWL) accompanying oxidative transformations of catecholamines (CA) into melanins, particularly adrenaline and noradrenaline in the model system CA + Fe(CN)6(3-) + OH(-) + H2O2 in vitro was investigated by spectroscopic methods. Separate steps of the oxidative transformations from CA to melanins were analyzed with respect to their energetic/spectroscopic properties in order to evaluate the possibility of chemiexcitation and light emission. Results of experiments with pure adrenochrome + H2O2 + OH- provided evidence pointing to the key role of the interaction between aminochromes and active oxygen species.