Identification of novel NAD(P)H dehydrogenase [quinone] 1 antagonist using computational approaches.

NAD(P)H: quinone oxidoreductase 1 (NQO1) inhibitors are proved as promising therapeutic agents against cancer. This study is to determine potent NAD(P)H-dependent NQO1 inhibitors with new scaffold. Pharmacophore-based three-dimensional (3D) QSAR model has been built based on 45 NQO1 inhibitors reported in the literature. The structure-function correlation coefficient graph represents the relationship between phase activity and phase predicted activity for training and test sets. A QSAR model statistics shows the excellent correlation of the generated model. Pharmacophore hypothesis (AARR) yielded a statistically significant 3D QSASR model with a correlation coefficient of r2 = 0.99 as well as an excellent predictive power. From the analysis of pharmacophore-based virtual screening using by SPEC database, 4093 hits were obtained and were further filtered using virtual screening filters (HTVS, SP, XP) through structure based molecular docking. Based on glide energy and docking score, seven lead compounds show better binding affinity compared to the co-crystal inhibitor. The results of induced fit docking and prime/MM-GBSA suggest that leads AN-153/J117103 and AT-138/KB09997 binding with the catalytic site. Further, to understanding the stability of identified lead compounds MD simulations were done. The lead AN-153/J117103 showed the strong binding stable of the protein-ligand complex. Also the computed drug likeness reveals potential of this compound to treat cancer. AbbreviationsNQO1NAD(P)H-quinine oxidoreductase 1CPHcommon pharmacophore hypothesisPLSpartial least squireHBDhydrogen bond donorSDstandard deviationXPextra precisionIFDinduced fit dockingMM-GBSAmolecular mechanics generalized born surface areaMDSmolecular dynamics simulationRMSDroot mean square deviationRMSFroot mean square fluctuationRMSEroot mean square errorADMEabsorption distribution metabolism excretionsCommunicated by Ramaswamy H. Sarma.

Heterobimetallic nickel(II) and palladium(II) complexes derived from S-benzyl-N- (ferrocenyl)methylenedithiocarbazate: Trypanocidal activity and interaction with Trypanosoma cruzi Old Yellow Enzyme (TcOYE).

Reactions of Ni(II) and Pd(II) precursors with S-benzyl-N-(ferrocenyl)methylenedithiocarbazate (HFedtc) led to the formation of heterobimetallic complexes of the type [MII(Fedtc)2] (M = Ni and Pd). The characterization of the compounds involved the determination of melting point, FTIR, UV-Vis, 1H NMR, elemental analysis and electrochemical experiments. Furthermore, the crystalline structures of HFedtc and [NiII(Fedtc)2] were determined by single crystal X-ray diffraction. The compounds were evaluated against the intracellular form of Trypanosoma cruzi (Tulahuen Lac-Z strain) and the cytotoxicity assays were assessed using LLC-MK2 cells. The results showed that the coordination of HFedtc to Ni(II) or Pd(II) decreases the in vitro trypanocidal activity while the cytotoxicity against LLC-MK2 cells does not change significantly. [PdII(Fedtc)2] showed the greater potential between the two complexes studied, showing an SI value of 8.9. However, this value is not better than that of the free ligand with an SI of 40, a similar value to that of the standard drug benznidazole (SI = 48). Additionally, molecular docking simulations were performed with Trypanosoma cruzi Old Yellow Enzyme (TcOYE), which predicted that HFedtc binds to the protein, almost parallel to the flavin mononucleotide (FMN) prosthetic group, while the [NiII(Fedtc)2] complex was docked into the enzyme binding site in a significantly different manner. In order to confirm the hypothetical interaction, in vitro experiments of fluorescence quenching and enzymatic activity were performed which indicated that, although HFedtc was not processed by the enzyme, it was able to act as a competitive inhibitor, blocking the hydride transfer from the FMN prosthetic group of the enzyme to the menadione substrate.

ADF and Cofilin1 Control Actin Stress Fibers, Nuclear Integrity, and Cell Survival.

Genetic co-depletion of the actin-severing proteins ADF and CFL1 triggers catastrophic loss of adult homeostasis in multiple tissues. There is impaired cell-cell adhesion in skin keratinocytes with dysregulation of E-cadherin, hyperproliferation of differentiated cells, and ultimately apoptosis. Mechanistically, the primary consequence of depleting both ADF and CFL1 is uncontrolled accumulation of contractile actin stress fibers associated with enlarged focal adhesions at the plasma membrane, as well as reduced rates of membrane protrusions. This generates increased intracellular acto-myosin tension that promotes nuclear deformation and physical disruption of the nuclear lamina via the LINC complex that normally connects regulated actin filaments to the nuclear envelope. We therefore describe a pathway involving the actin-severing proteins ADF and CFL1 in regulating the dynamic turnover of contractile actin stress fibers, and this is vital to prevent the nucleus from being damaged by actin contractility, in turn preserving cell survival and tissue homeostasis.

Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP(+)-dependent dehydrogenases of the pentose phosphate pathway.

Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP(+)-dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibited the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI50: 1.5 to 20µM) and caused a significant (p<0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg(2+)>MeHg≈EtHg>TM (p<0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system.

Critical roles for multiple formins during cardiac myofibril development and repair.

Cardiac and skeletal muscle function depends on the proper formation of myofibrils, which are tandem arrays of highly organized actomyosin contractile units called sarcomeres. How the architecture of these colossal molecular assemblages is established during development and maintained over the lifetime of an animal is poorly understood. We investigate the potential roles in myofibril formation and repair of formin proteins, which are encoded by 15 different genes in mammals. Using quantitative real-time PCR analysis, we find that 13 formins are differentially expressed in mouse hearts during postnatal development. Seven formins immunolocalize to sarcomeres in diverse patterns, suggesting that they have a variety of functional roles. Using RNA interference silencing, we find that the formins mDia2, DAAM1, FMNL1, and FMNL2 are required nonredundantly for myofibrillogenesis. Knockdown phenotypes include global loss of myofibril organization and defective sarcomeric ultrastructure. Finally, our analysis reveals an unanticipated requirement specifically for FMNL1 and FMNL2 in the repair of damaged myofibrils. Together our data reveal an unexpectedly large number of formins, with diverse localization patterns and nonredundant roles, functioning in myofibril development and maintenance, and provide the first evidence of actin assembly factors being required to repair myofibrils.

NADPH-d and Fos reactivity in the rat spinal cord following experimental spinal cord injury and embryonic neural stem cell transplantation.

AIMS: The aim of this study is to determine the role of nitric oxide (NO) in neuropathic pain and the effect of embryonic neural stem cell (ENSC) transplantation on NO content in rat spinal cord neurons following spinal cord injury (SCI). MAIN METHODS: Ninety adult male Sprague-Dawley rats were divided into 3 groups (n=30, each): control (laminectomy), SCI (hemisection at T12-T13 segments) and SCI+ENSC. Each group was further divided into sub-groups (n=5 each) based on the treatment substance (L-NAME, 75 mg/kg/i.p.; L-arginine, 225 mg/kg/i.p.; physiological saline, SF) and duration (2h for acute and 28 days for chronic groups). Pain was assessed by tail flick and Randall-Selitto tests. Fos immunohistochemistry and NADPH-d histochemistry were performed in segments 2 cm rostral and caudal to SCI. KEY FINDINGS: Tail-flick latency time increased in both acute and chronic L-NAME groups and increased in acute and decreased in chronic L-arginine groups. The number of Fos (+) neurons decreased in acute and chronic L-NAME and decreased in acute L-arginine groups. Following ENSC, Fos (+) neurons did not change in acute L-NAME but decreased in the chronic L-NAME groups, and decreased in both acute and chronic L-arginine groups. NADPH-d (+) neurons decreased in acute L-NAME and increased in L-arginine groups with and without ENSC transplantation. SIGNIFICANCE: This study confirms the role of NO in neuropathic pain and shows an improvement following ENSC transplantation in the acute phase, observed as a decrease in Fos(+) and NADPH-d (+) neurons in spinal cord segments rostral and caudal to injury.

Dynamic interaction of formin proteins and cytoskeleton in mouse oocytes during meiotic maturation.

Formin-2 (Fmn2) nucleates actin filaments required for spindle migration during the metaphase of meiosis I in mouse oocytes. While recent studies showed that Fmn2 is involved in the formation of a dynamic actin meshwork on meiotic spindle and the migration of chromosomes, the precise location and the mechanism of action of Fmn2 in the mouse oocyte is not known. In this work, we show that Fmn2 is colocalized with spindle during metaphase I (MI) and this pattern is lost in nocodazole-treated oocytes. Fmn2 directly interacts with polymerized microtubules (MTs) in vitro via a well-conserved domain called formin homology 2 (FH2). Microinjection of mRNA encoding formin homology 1 (FH1)FH2 domains of Fmn2 into Fmn2-/- oocytes partially rescued the defect of polar body extrusion, while mRNAs encoding FH2 domain alone could not rescue the defect. mDia1 and mDia2, Diaphanous (Dia) subfamily of formin proteins, exhibit unique patterns of expression in mouse oocytes. While mDia1 is localized on meiotic spindle, mDia2 localization is confined in spindle poles similar to γ-tubulin. Collectively, our results suggest that the ability of Fmn2 to directly interact with MTs and to polymerize actins via the conserved FH1FH2 domains is crucial for chromosomal migration in MI oocytes. We also show that mDia1 and mDia2 are dynamic components of meiotic spindle and pole complex during meiotic maturation of oocytes.

Structure of the inhibitor complex of old yellow enzyme from Trypanosoma cruzi.

Old yellow enzyme (OYE) is an NADPH oxidoreductase which contains flavin mononucleotide as prosthetic group. The X-ray structures of OYE from Trypanosoma cruzi (TcOYE) which produces prostaglandin (PG) F(2α) from PGH(2) have been determined in the presence or absence of menadione. The binding motif of menadione, known as one of the inhibitors for TcOYE, should accelerate the structure-based development of novel anti-chagasic drugs that inhibit PGF(2α) production specifically.

Biocatalysis with thermostable enzymes: structure and properties of a thermophilic 'ene'-reductase related to old yellow enzyme.

We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

Isoform-selective chemical inhibition of mDia-mediated actin assembly.

Formins are potent actin assembly factors. Diaphanous formins, including mDia1, mDia2, and mDia3 in mammals, are implicated in mitosis and cytokinesis, but no chemical interactors have been reported. We developed an in vitro screen for inhibitors of actin assembly by mDia1 and identified an inhibitor of mDia1 and mDia2 that does not inhibit mDia3 at the concentrations tested. These results establish the druggability of mDia formins and introduce a first-generation inhibitor.

NAD(P)H quinone oxidoreductase 1 is essential for ozone-induced oxidative stress in mice and humans.

One host susceptibility factor for ozone identified in epidemiologic studies is NAD(P)H quinone oxidoreductase 1 (NQO1). We hypothesized that after ozone exposure, NQO1 is required to increase 8-isoprostane (also known as F(2)-isoprostane) production, a recognized marker of ozone-induced oxidative stress, and to enhance airway inflammation and hyperresponsiveness. In this report, we demonstrate that in contrast to wild-type mice, NQO1-null mice are resistant to ozone and have blunted responses, including decreased production of F(2)-isoprostane and keratinocyte chemokine, decreased airway inflammation, and diminished airway hyperresponsiveness. Importantly, these results in mice correlate with in vitro findings in humans. In primary human airway epithelial cells, inhibition of NQO1 by dicumarol blocks ozone-induced F(2)-isoprostane production and IL-8 gene expression. Together, these results demonstrate that NQO1 modulates cellular redox status and influences the biologic and physiologic effects of ozone.

[Wheat root cells functioning under inhibition of I and II complexes of mitochondrial respiratory chain].

A joint effect of rotenone and malonate on the intensity of respiration, output of K+ and ultrastructure of wheat root cells treated for 6 h was studied. The addition of malonate to rotenone containing solution, in which wheat roots had been incubated for an hour, caused further decrease in respiration intensity and K+ output into external medium. Many mitochondria acquired torus shape in 2h after malonate addition. The increase in respiratory intensity and re-entry of K+ from the incubation medium into the cells were observed during following hours of incubation. We assume that reparation and adaptation processes took place in this case. The observed contacts of endoplasmic reticulum lumens with mitochondria are indicative of possible synthesis of an enzyme able to metabolize malonate to acetyl-CoA and CO2. We propose that torus shape of mitochondria is due to the increase in their outer surfaces, that, in turn, is a result of activation of external NAD(P)H-dehydrogenase. These findings may be evidence of possible adaptation of the root cells to the joint effect of the inhibitors.

Regulation of common carotid arterial blood flow by nitrergic neurons in the medulla of cats.

Glutamate stimulation of the dorsal facial area, an area located dorsal to the facial nucleus, increases common carotid arterial blood flow. Nitrergic neurons are important in cardiovascular regulatory areas. We investigated whether the nitrergic neurons might be present and play a role in the dorsal facial area to regulate the arterial blood flow. Injections of L-arginine (an NO precursor) and sodium nitroprusside (an NO donor) into the area caused dose-dependent increases in the arterial blood flow. Injection of N(G)-nitro-arginine methyl ester (L-NAME, an NO synthase inhibitor) or methylene blue (a guanylate cyclase inhibitor) decreased the arterial blood flow. Nitrergic neurons and fibers were found in the dorsal facial area by histochemical staining of nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase, a maker of NO synthase. In conclusion, nitrergic neurons are present in the dorsal facial area and appear to release NO tonically in stimulating the area to cause increase in common carotid arterial blood flow.

Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells.

Oxidative stress may be an important determinant of the severity of acute pancreatitis. One-electron reduction of oxidants generates reactive oxygen species (ROS) via redox cycling, whereas two-electron detoxification, e.g. by NAD(P)H:quinone oxidoreductase, does not. The actions of menadione on ROS production and cell fate were compared with those of a non-cycling analogue (2,4-dimethoxy-2-methylnaphthalene (DMN)) using real-time confocal microscopy of isolated perfused murine pancreatic acinar cells. Menadione generated ROS with a concomitant decrease of NAD(P)H, consistent with redox cycling. The elevation of ROS was prevented by the antioxidant N-acetyl-l-cysteine but not by the NADPH oxidase inhibitor diphenyliodonium. DMN produced no change in reactive oxygen species per se but significantly potentiated menadione-induced effects, probably via enhancement of one-electron reduction, since DMN was found to inhibit NAD(P)H:quinone oxidoreductase detoxification. Menadione caused apoptosis of pancreatic acinar cells that was significantly potentiated by DMN, whereas DMN alone had no effect. Furthermore, bile acid (taurolithocholic acid 3-sulfate)-induced caspase activation was also greatly increased by DMN, whereas DMN had no effect per se. These results suggest that acute generation of ROS by menadione occurs via redox cycling, the net effect of which is induction of apoptotic pancreatic acinar cell death. Two-electron detoxifying enzymes such as NAD(P)H:quinone oxidoreductase, which are elevated in pancreatitis, may provide protection against excessive ROS and exert an important role in determining acinar cell fate.

Detection of NADPH-diaphorase activity in Paramecium primaurelia.

Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell-cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for beta-NADPH and alpha-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized. The non-denaturing electrophoresis, followed by exposure to beta-NADPH or alpha-NADH co-substrates, revealed the presence of a band of apparent molecular mass of about 124 kDa or a band of apparent molecular mass of about 175 kDa, respectively. In immunoblot experiments, the bNOS antibody recognized a single band of apparent molecular mass of about 123 kDa.

The basic region of the diaphanous-autoregulatory domain (DAD) is required for autoregulatory interactions with the diaphanous-related formin inhibitory domain.

Mammalian diaphanous-related (mDia) formins act as Rho GTPase effectors during cytoskeletal remodeling. Rho binding to mDia amino-terminal GTPase-binding domains (GBDs) causes the adjacent Dia-inhibitory domain (DID) to release the carboxyl-terminal Dia-autoregulatory (DAD) domain that flanks the formin homology-2 (FH2) domain. The release of DAD allows the FH2 domain to then nucleate and elongate nonbranched actin filaments. DAD, initially discovered as a region of homology shared between a phylogenetically divergent set of formin proteins, is comprised of a core motif, MDXLLXL, and an adjacent region is comprised of numerous basic residues, typically RRKR in the mDia family. Here, we show that these specific amino acids within the basic region of DAD contribute to the binding of DID and therefore the maintenance of the mDia autoregulatory mechanism. In addition, expression of full-length versions of mDia2 containing amino acid substitutions in either the DAD core or basic regions causes profound changes in the F-actin architecture, including the formation of filopodia-like structures that rapidly elongate from the cell edge. These studies further refine our understanding of the molecular contribution of DAD to mDia control and the role of mDia2 in the assembly of membrane protrusions.

Extremely potent triterpenoid inducers of the phase 2 response: correlations of protection against oxidant and inflammatory stress.

A series of synthetic triterpenoid (TP) analogues of oleanolic acid are powerful inhibitors of cellular inflammatory processes such as the induction by IFN-gamma of inducible nitric oxide synthase (iNOS) and of cyclooxygenase 2 in mouse macrophages. Here, we show that these analogues are also extremely potent inducers of the phase 2 response [e.g., elevation of NAD(P)H-quinone oxidoreductase and heme oxygenase 1], which is a major protector of cells against oxidative and electrophile stress. Moreover, like previously identified phase 2 inducers, the TP analogues use the antioxidant response element-Nrf2-Keap1 signaling pathway. Thus, induction of the phase 2 response and suppression of the iNOS induction was abrogated in nrf2(-/-) and keap1(-/-) mouse embryonic fibroblasts. The high potency of TP analogues in inducing the phase 2 response and blocking inflammation depends on the presence of activated Michael reaction (enone) functions at critical positions in rings A and C. The most potent TP doubles NAD(P)H-quinone oxidoreductase in murine hepatoma cells at 0.28 nM and has an IC(50) for suppression of iNOS induction in primary mouse macrophages of 0.0035 nM. The direct interaction of this TP with thiol groups of the Keap1 sensor for inducers is demonstrated spectroscopically. The antiinflammatory and phase 2 inducer potencies of 18 TP are closely linearly correlated (r(2) = 0.91) over 6 orders of magnitude of concentration. Thus, in addition to blocking inflammation and promoting differentiation, these TP exhibit another very important protective property: the induction of the phase 2 response.

Site-specific inhibitors of NADPH oxidase activity and structural probes of flavocytochrome b: characterization of six monoclonal antibodies to the p22phox subunit.

The integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the human phagocyte NADPH oxidase, an enzyme complex that initiates a cascade of reactive oxygen species important in the elimination of infectious agents. This study reports the generation and characterization of six mAbs (NS1, NS2, NS5, CS6, CS8, and CS9) that recognize the p22(phox) subunit of the Cyt b heterodimer. Each of the mAbs specifically detected p22(phox) by Western blot analysis but did not react with intact neutrophils in FACS studies. Phage display mapping identified core epitope regions recognized by mAbs NS2, NS5, CS6, CS8, and CS9. Fluorescence resonance energy transfer experiments indicated that mAbs CS6 and CS8 efficiently compete with Cascade Blue-labeled mAb 44.1 (a previously characterized, p22(phox)-specific mAb) for binding to Cyt b, supporting phage display results suggesting that all three Abs recognize a common region of p22(phox). Energy transfer experiments also suggested the spatial proximity of the mAb CS9 and mAb NS1 binding sites to the mAb 44.1 epitope, while indicating a more distant proximity between the mAb NS5 and mAb 44.1 epitopes. Cell-free oxidase assays demonstrated the ability of mAb CS9 to markedly inhibit superoxide production in a concentration-dependent manner, with more moderate levels of inhibition observed for mAbs NS1, NS5, CS6, and CS8. A combination of computational predictions, available experimental data, and results obtained with the mAbs reported in this study was used to generate a novel topology model of p22(phox).

Cortical interneurons become activated by deafferentation and instruct the apoptosis of pyramidal neurons.

Unlike peripheral nervous system neurons and certain groups of nerve cells in the CNS, cortical projection neurons are tolerant of axonal lesions. This resistance is incongruent with the massive death of pyramidal neurons in age-associated neurodegenerative diseases that proceed along corticocortical connections. Some insights have emerged from our previous work showing that pyramidal cells in piriform cortex undergo classical apoptosis within 24 h after bulbectomy via transsynaptic, but not retrograde, signaling. These findings allow the investigation of cellular and molecular changes that take place in the context of experimental cortical degeneration. In the present study, we show that the transsynaptic death of pyramidal neurons in piriform cortex is a nitric oxide-mediated event signaled by activated interneurons in layer I. Thus, we demonstrate that cortical interneurons play an essential role in transducing injury to apoptotic signaling that selectively targets pyramidal neurons. We propose that this mechanism may be generic to cortical degenerations and amenable to therapeutic interventions.

Homocysteine enhances TIMP-1 expression and cell proliferation associated with NADH oxidase in rat mesangial cells.

BACKGROUND: Recent studies in our laboratory demonstrated that chronic hyperhomocysteinemia (hHcys) induced glomerular sclerosis. The mechanism mediating hHcys-induced glomerular damage remains unknown. The present study was designed to test a hypothesis that homocysteine (Hcys) increases the production by nicotinamide adenine dinucleotide (NADH) oxidase and thereby stimulates the formation of tissue inhibitor of metalloproteinase (TIMP-1) in rat mesangial cells, consequently leads to glomerulosclerosis. METHODS: Rat mesangial cells were incubated with L-homocysteine (L-Hcys) to determine the effects of Hcys on cell proliferation and metabolism of extracellular matrix (ECM). Northern blot, Western blot, oligonucleotide transfection, measurements of NADH oxidase activity and levels, and cell proliferation assay were performed. RESULTS: In cultured rat mesangial cells, treatment with L-Hcys (40 to 160 micromol/L) markedly increased the mRNA levels of TIMP-1 and Gp91 and led to accumulation of collagen I, which were accompanied by enhanced cell proliferation and NADH oxidase activity in mesangial cells. These Hcys-induced biochemical and functional changes were substantially blocked by a NADH oxidase inhibitor, diphenylene iodonium chloride (DPI) or a superoxide dismutase (SOD) mimetic, hydroxyl-tetramethylpiperidin-oxyl (TEMPOL). Moreover, blockade of NADH oxidase subunit, phox22, by its antisense oligodeoxynucleotide also eliminated the increase in NADH oxidase activity induced by L-Hcys. CONCLUSION: These results indicate that Hcys-induced alterations of ECM metabolism in mesangial cells are associated with enhanced NADH oxidase activity and that oxidative stress-stimulated up-regulation of TIMP-1 may play an important role in the deposition of collagen or ECM elements in the glomeruli during hHcys.