Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.3.1 (NADPH oxidase)
 11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

While acute lindane treatment and chronic ethanol feeding to rats have been associated with hepatic oxidative stress, the possible roles of these stresses in the pathogenesis of hepatic lesions reported in acute lindane intoxication and in those observed in some models of chronic alcoholism have not been established. Our previous studies in rats chronically fed ethanol regimens and then treated with a single intraperitoneal (i.p.) dose of lindane (20 mg/kg) showed that while lindane per se was invariably associated with hepatic oxidative stress, chronic ethanol feeding only produced this stress when the dietary level of vitamin E was relatively low. Chronic ethanol pretreatment did not significantly affect the lindane-associated oxidative stress, and neither chronic ethanol feeding nor acute lindane, single or in combination, produced any histologic and biochemical evidence of liver damage. In the present experiment, the acute dose of lindane was increased to 40 mg/kg, and we have studied a larger number of prooxidant and antioxidant hepatic factors. Male Wistar rats (115.5 +/- 5.4 g) were fed ad lib for 11 weeks a calorically well-balanced and nutritionally adequate basal diet, or the same basal diet plus a 32% ethanol/25% sucrose solution, also ad lib, and were then injected i.p. with a single dose of lindane or with equivalent amounts of corn oil. The results indicated that acute lindane treatment to naive rats increased practically all the prooxidant hepatic factors examined (cytochromes P450 and b5, NADPH cytochrome c reductase, NADPH oxidase), as well as the generation of microsomal superoxide radical and thiobarbituric acid reactive substances of liver homogenates, but did not modify any of the antioxidant hepatic factors studied. Conversely, the chronic administration of ethanol alone did not significantly affect the prooxidant hepatic factors but reduced some of the antioxidants (i.e., the activities of GSH-Px and the contents of alpha-tocopherol and ubiquinols 9 and 10). Although chronic ethanol pretreatment further increased the superoxide generation induced by lindane per se, it did not increase but generally reduced the effects of lindane per se on the other prooxidant factors studied. Furthermore, although acute lindane administration to ethanol-pretreated rats was associated with decreases in GSH and catalase (not affected by ethanol or lindane treatment alone), it did not substantially modify the reducing effects of ethanol feeding per se on GSH-Px, alpha-tocopherol, and ubiquinols. Once again, neither chronic ethanol feeding nor lindane treatment, single or in combination, was associated with any evidence of liver damage.
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PMID:Prooxidant and antioxidant hepatic factors in rats chronically fed an ethanol regimen and treated with an acute dose of lindane. 754 17

Nitric oxide (NO) reacts with heme-containing enzymes, including certain isoforms of cytochrome P450. Cytochrome P4502E1 (CYP2E1) is induced by ethanol and plays an important role in the toxicity of ethanol and other hepatotoxins. CYP2E1 is also very effective in generating reactive oxygen intermediates such as superoxide radical and H2O2, oxidizing ethanol to the 1-hydroxyethyl radical, and has a high NADPH oxidase activity. The effect of NO on CYP2E1 catalytic activity and generation of reactive oxygen intermediates was evaluated. Incubating liver microsomes isolated from rats treated with pyrazole to induce high levels of CYP2E1, with gaseous NO or NO released from a variety of NO donors such as SNAP, DEA/NO, spermine/NO, and GSNO, resulted in a loss of CYP2E1 catalytic activity with specific substrates such as p-nitrophenol or dimethylnitrosamine. Trapping of NO with hemoglobin resulted in protection of CYP2E1 activity against the inactivation by NO. There was no effect by analogues of the donors which do not release NO nor was there any effect by NO on NADPH-cytochrome P450 reductase activity. Inactivation of CYP2E1 by NO was not prevented by superoxide dismutase or catalase, suggesting that superoxide, H2O2, or peroxynitrite were not responsible for the actions of NO. The inactivated CYP2E1 was not degraded nor did it lose its epitope sites as shown by Western blot analysis. Associated with loss of CYP2E1 catalytic activity was a decrease in the formation of superoxide radical and H2O2, in microsomal lipid peroxidation catalyzed by low, but not high concentration of iron, and in consumption of NADPH. Oxidation of ethanol to the 1-hydroxyethyl radical was also inhibited by NO. ESR experiments indicated the formation of stable heme-NO complexes with CYP2E1. NO appears to compete with O2 and CO for binding to CYP2E1 as incubation with gaseous NO, or NO donors inhibited formation of the characteristic CO binding spectrum of P450. Microsomes isolated from a stably transfected HepG2 cell line expressing only CYP2E1 were also inactivated by NO, validating interaction of NO with this isoform of P450. These results indicate that NO inhibits CYP2E1 catalytic activity and generation of reactive radical intermediates. NO may prevent toxicity of agents which require bioactivation by P450 isoforms such as CYP2E1 and in generation of reactive intermediates by CYP2E1.
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PMID:Inhibition of rat and human cytochrome P4502E1 catalytic activity and reactive oxygen radical formation by nitric oxide. 901 19

The following describes a novel screening method for "new chemical entities" (NCEs), suitable for ADMET studies, that measures ability to form prooxidant radicals on metabolism and their ability to induce oxidative stress in intact cells. The accelerated molecular cytotoxic mechanism screening (ACMS) techniques used with isolated rat hepatocytes showed that cytotoxicity is usually initiated as a result of macromolecular covalent binding or macromolecular oxidative stress. While P450 is likely responsible for drug metabolic activation in the liver, intestine, lung, and in other nonhepatic tissues, where P450 levels are low, peroxidases including prostaglandin synthetase peroxidase can catalyze xenobiotic one-electron oxidation to form prooxidant free radicals that may cause toxicity or carcinogenesis. Inflammation markedly activates H2O2, generating NADPH oxidase and peroxidase of certain immune cells when they infiltrate tissues including the liver. Myeloperoxidase and NADPH oxidase in the Kupffer cells (resident macrophages of the liver) also become activated during inflammation. The addition of noncytotoxic concentrations of peroxidase/H2O2 to the hepatocyte incubate markedly increased drug cytotoxicity and prooxidant radical formation as shown by glutathione or lipid oxidation. Many drugs that have hepato- or gastrointestinal (GI) toxicity problems or were withdrawn from the market for safety problems, e.g., troglitazone, tolcapone, mefenamic acid, diclofenac, and phenylbutazone, were markedly more toxic and prooxidant in this inflammation model system, whereas other drugs, e.g., entacapone, were not toxic in this inflammation model. Some of the idiosyncratic hepatotoxicity responsible for recent drug withdrawals may therefore result from commonplace sporadic inflammatory episodes during drug therapy.
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PMID:Oxidative stress mediated idiosyncratic drug toxicity. 1593 67

Mitochondrial P450 type enzymes catalyze central steps in steroid biosynthesis, including cholesterol conversion to pregnenolone, 11beta and 18 hydroxylation in glucocorticoid and mineralocorticoid synthesis, C-27 hydroxylation of bile acids, and 1alpha and 24 hydroxylation of 25-OH-vitamin D. These monooxygenase reactions depend on electron transfer from NADPH via FAD adrenodoxin reductase and 2Fe-2S adrenodoxin. These systems can function as a futile NADPH oxidase, oxidizing NADPH in absence of substrate, and leak electrons via adrenodoxin and P450 to O(2), producing superoxide and other reactive oxygen species (ROS). The degree of uncoupling depends on the P450 and steroid substrate. Studies with purified proteins and overexpression in cultured cells show consistently that adrenodoxin, but not reductase, is responsible for ROS production that can lead to apoptosis. In the ovary and corpus luteum, antioxidant enzyme activities superoxide dismutase, catalase, and glutathione peroxidase parallel steroidogenesis. Antioxidant beta-carotene, alpha-tocopherol, and ascorbate can protect against oxidative damages of P450 systems. In testis Leydig cells, steroidogenesis is associated with aging of the steroidogenic capacity.
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PMID:Antioxidant protective mechanisms against reactive oxygen species (ROS) generated by mitochondrial P450 systems in steroidogenic cells. 1668 56

Long-term treatment of hypertensive disorders with hydralazine has resulted in some patients developing hepatitis and lupus erythematosus, an autoimmune syndrome. The concentration of hydralazine required to cause 50% cytotoxicity in 2 h (LC(50)) toward isolated rat hepatocytes was found to be 8 mM. Cytotoxicity was delayed by the P450 inhibitor, 1-aminobenzotriazole, suggesting that P450 catalyzed the formation of toxic metabolites from hydralazine. No hydralazine-induced oxidative stress was apparent as there was little effect on hepatocyte lipid peroxidation, protein carbonyl formation, intracellular H(2)O(2), or hepatocyte GSH levels and no effect of butylated hydroxyanisole (BHA) on cytotoxicity. Drug-induced hepatotoxicity in vivo has often been attributed to infiltrating inflammatory cells, for example, neutrophils or resident Kupffer cells whose NADPH oxidase generates H(2)O(2), when activated. The effect of a nontoxic continuous infusion of H(2)O(2) on hydralazine cytotoxicity was investigated. It was found that H(2)O(2) increased hepatocyte susceptibility to hydralazine 4-fold (LC(50), 2 mM). Cytotoxicity was still prevented by the P450 inhibitor but now involved some oxidative stress as shown by increased protein carbonyls, endogenous H(2)O(2), and GSH oxidation. Lipid peroxidation was not increased, and cytotoxicity was not inhibited by BHA. Cytotoxicity, however, was inhibited by 4-hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (TEMPOL), a ROS scavenger. Because neutrophils or Kupffer cells release myeloperoxidase on activation, the effect of adding peroxidase to the hepatocytes exposed to H(2)O(2) on hydralazine cytotoxicity was investigated. It was found that peroxidase/H(2)O(2) increased hepatocyte susceptibility to hydralazine 80-fold (LC 50, 0.1 mM). Furthermore, cytotoxicity occurred following extensive oxidative stress that included lipid peroxidation, and cytotoxicity that was now prevented by the antioxidant BHA. These results indicate that three cytotoxic pathways exist for hydralazine: a P450-catalyzed pathway not involving oxidative stress, a P450/H(2)O(2)-catalyzed oxidative stress-mediated cytotoxic pathway not involving lipid peroxidation, and a peroxidase/H(2)O(2)-catalyzed lipid peroxidation-mediated cytotoxic pathway.
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PMID:Accelerated cytotoxic mechanism screening of hydralazine using an in vitro hepatocyte inflammatory cell peroxidase model. 1839 51

An early increase in ROS production is characteristic of cerebellar granule cells undergoing apoptosis in the presence of 5 mM KCl. However, the sources of this increase have not been investigated in detail. In particular whether there is a single enzymatic source or the increase in ROS production is the consequence of the involvement of different enzymes has not been studied in depth. Different enzymatic pathways may indeed contribute to the up-regulation of intracellular ROS production either directly or via side-chain reactions and a number of candidate enzymes are known to be involved in the apoptotic process in various cell types. The aim of this study was to identify the cellular sources of the ROS generated by CGCs undergoing apoptosis by low K+. A panel of specific inhibitors against phospholipase, cytochromes P450, cyclooxygenase, lipoxygenase, xanthine oxidase, ribonucleotide reductase and NADPH oxidase were used. We provide evidence that no single source of ROS can be identified in apoptotic CGCs, but the ROS generated through the arachidonic acid (AA) pathways, mainly via lipoxygenase activities, seems to be the most prominent.
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PMID:Different sources of reactive oxygen species contribute to low potassium-induced apoptosis in cerebellar granule cells. 1850 67

Measuring NADPH oxidase (Nox)-derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH-stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1-Nox2-Nox4-/-) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH-stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH-stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH-stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH-dependent signal in assays utilizing lucigenin, L-012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR-/-) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR-/- abolished the signal in NADPH-stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells treated with angiotensin II showed an increased NADPH-dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox.
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PMID:Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays. 2786 90

In non-alcoholic steatohepatitis (NASH), hepatic stellate cells (HSC) differentiate into myofibroblast-like cells that cause fibrosis, which predisposes patients to cirrhosis and hepatocellular carcinoma. Thus, modeling interactions between activated HSCs and hepatocytes in vitro can aid in the development of anti-NASH/fibrosis therapeutics and lead to a better understanding of disease progression. Species-specific differences in drug metabolism and disease pathways now necessitate the supplementation of animal studies with data acquired using human liver models; however, current models do not adequately model the negative effects of primary human activated HSCs on the phenotype of otherwise well-differentiated primary human hepatocytes (PHHs) as in vivo. Therefore, here we first determined the long-term effects of primary human activated HSCs on PHH phenotype in a micropatterned co-culture (MPCC) platform while using 3T3-J2 murine embryonic fibroblasts as the control cell type since it has been shown previously to stabilize PHH functions for 4-6 weeks. We found that HSCs were not able to stabilize the PHH phenotype to the same magnitude and longevity as the fibroblasts, which subsequently inspired the development of a micropatterned tri-culture (MPTC) platform in which (a) micropatterned PHHs were functionally stabilized using fibroblasts, and (b) the PHH phenotype was modulated by culturing HSCs within the fibroblast monolayer at physiologically-relevant ratios with PHHs. Transwell inserts containing HSCs were placed atop MPCCs containing fibroblasts to confirm the effects of paracrine signaling between PHHs and HSCs. We found that while albumin and urea secretions were relatively similar in MPTCs and MPCCs (suggesting well-differentiated PHHs), increasing HSC numbers within MPTCs downregulated hepatic cytochrome-P450 (2A6, 3A4) and transporter activities, and caused steatosis over 2 weeks. Furthermore, MPTCs secreted higher levels of pro-inflammatory interleukin-6 (IL-6) cytokine and C-reactive protein (CRP) than MPCCs. Treatment of MPCCs with HSC-conditioned culture medium confirmed that HSC secretions mediate the altered phenotype of PHHs observed in MPTCs, partly via IL-6 signaling. Lastly, we found that NADPH oxidase (NOX) inhibition and farnesoid X receptor (FXR) activation using clinically relevant drugs alleviated hepatic dysfunctions in MPTCs. In conclusion, MPTCs recapitulate symptoms of NASH- and early fibrosis-like dysfunctions in PHHs and have utility for drug discovery in this space.
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PMID:Microengineered cultures containing human hepatic stellate cells and hepatocytes for drug development. 2870 67

Acetaminophen (APAP) overdose is the most frequent cause of liver injury and acute liver failure in many western countries. The mechanism of APAP-induced hepatocyte necrosis has been investigated extensively. The formation of a reactive metabolite and its binding to cellular proteins was initially thought to be responsible for cell death. A competing hypothesis was introduced that questioned the relevance of protein binding and instead suggested that P450-derived oxidant stress and lipid peroxidation causes APAP-induced liver injury. However, work over the last 15 years has reconciled some of these apparent contradictory hypotheses. This review summarizes the present state of knowledge on the role of reactive oxygen species (ROS) in APAP hepatotoxicity. Detailed investigations into the sources and relevance of the oxidant stress have clearly shown the critical role of the electron transport chain of mitochondria as main source of the oxidant stress. Other potential sources of ROS such as cytochrome P450 enzymes or NADPH oxidase on phagocytes are of limited relevance. The mitochondria-derived superoxide and peroxynitrite formation is initiated by the binding of the reactive metabolite to mitochondrial proteins and the amplification by mitogen activated protein kinases. The consequences of this oxidant stress are the opening of the mitochondrial membrane permeability transition pore with cessation of ATP synthesis, nuclear DNA fragmentation and ultimately cell necrosis. Lipid peroxidation is not a relevant mechanism of cell death but can be a marker of ROS formation. These mechanistic insights suggest that targeting mitochondrial oxidant stress is a promising therapeutic option for APAP hepatotoxicity.
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PMID:Oxidant Stress and Lipid Peroxidation in Acetaminophen Hepatotoxicity. 2968 14