Stimulated Reactive Oxygen Species Formation Research Articles

Gingival crevicular fluid from pregnant women impairs trophoblast cell function and trophoblast‐neutrophil interaction

A strong association between periodontitis and higher susceptibility to pregnancy complications like preeclampsia has been reported although the mechanisms remain elusive. Trophoblast cells modulate the recruitment and functional shaping of maternal leukocytes at early stages to sustain an antiinflammatory microenvironment and fetal growth. Neutrophil activation with reactive oxygen species (ROS) release is associated with preeclampsia. Our aim was to study the effect of the gingival crevicular fluid (GCF) from pregnant women on trophoblast cell function and trophoblast-neutrophil interaction. Pregnant women at 16-20weeks of gestation (n=27) and non-pregnant women (n=8) as the control group were studied for gingivoperiodontal clinical score evaluation and GCF collection. Total bacteria and common periodontal pathogens were analyzed in GCF samples. The effect of each GCF sample was tested on first trimester trophoblast-derived cells to assess cell migration, cytokine expression and glucose uptake. Also, the effect of GCF on human peripheral neutrophil chemoattraction by trophoblast cells and ROS formation was assessed. Gingival crevicular fluid from pregnant women reduced trophoblast cell migration, increased proinflammatory marker expression and glucose uptake. A significant correlation between gingivoperiodontal score and trophoblast dysfunction was observed. Upon conditioning of trophoblast cells with GCF, only the GCF from pregnant women stimulated neutrophil chemoattraction. Similarly, GCF from pregnant but not from non-pregnant controls stimulated ROS formation in neutrophils. Gingival crevicular fluid from pregnant women is deleterious for first trimester trophoblast cell function. These effects could lead to placental homeostasis disruption underlying a pathogenic mechanism of pregnancy complications associated to periodontal disease.

Isolation and expression analysis of cadmium-induced genes from Cd/Mn hyperaccumulator Phytolacca americana inresponse to high Cd exposure.

Phytolacca americana is recognised as a hyperaccumulator that accumulates cadmium (Cd) and manganese (Mn). Although most studies have provided abundant physiological evidence, little is known about the molecular mechanisms of Cd accumulation in P.americana. In this study, Cd-induced genes were isolated using suppression subtractive hybridisation (SSH) library construction, and gene expression patterns under Cd stress were quantified using real-time quantitative PCR. The functions of PaGST, PaMT and PaFe-SOD were confirmed in transformant yeast. Reactive oxygen species (ROS) formation and cell death in root tips were detected, and SOD and POD activities in leaf tissue were also analysed. There were about 447 expressed sequence tags (ESTs) identified and confirmed. GO analysis showed those genes were mainly involved in metabolism, cell stress and defence, transcription and translation, signal transduction, transport, energy and ion transport, which formed the basis for a molecular understanding of P.americana Cd tolerance mechanisms. Cd also stimulated ROS formation and modified the antioxidant systems. Taken together, our results indicate that ROS formation and Cd-induced gene expression favour P.americana tolerance by activating the defence system and permitting subsequent adaptation to Cd toxicity.

Mir-29b Regulates Oxidative Stress by Targeting SIRT1 in Ovarian Cancer Cells

Background: Metabolic abnormalities are frequently observed in multiple malignancies including epithelial ovarian cancer (EOC), among which imbalance between generation and elimination of reactive oxygen species (ROS) plays a critical role in EOC onset and progression. Here we investigated the role of miR-29b, a well-established tumor-suppressor miRNA in metabolic regulation of EOC cells. Methods: cell viability and apoptosis in miR-29b inhibited and over-expressed EOC cells were evaluated by CCK8 and Annexin V–FITC/PI assays. Change in miR-29b was detected in EOC cells incubated in H₂O₂ culture by q-PCR. Relative ROS levels were also detected in different EOC cultures, including modified miR-29b and SIRT1 levels as well as H₂O₂ incubation. A luciferase reporter assay was employed to detect the direct binding of miR-29b to SIRT1 3’ UTR. Changes in cell viability and ROS levels were assessed in SIRT1-knocked down EOC cells. Results: miR-29b expression correlates with decreased EOC cell viability and increased apoptosis. H₂O₂ downregulated miR-29b in a time and dose-dependent manner. miR-29b expression negatively correlated with ROS levels, whereas SIRT1 significantly stimulated ROS formation. Luciferase reporter assays confirmed miR-29b downregulation of SIRT1by directly targeting its mRNA 3’-UTR. SIRT1 silencing rescues cell viability of H₂O₂ treated cells. Also, SIRT1 inhibition blocked cell apoptosis induced by H₂O₂ as well as reduced intracellular ROS levels. Conclusion: Together, our findings indicated that the miR-29b/SIRT1 axis has a protective effect against H₂O₂-induced damage of cell viability and oxidative stress and may provide novel options for miR-29b-based therapeutic approaches for EOC treatment.

Role of c-Met/Phosphatidylinositol 3-Kinase (PI3k)/Akt Signaling in Hepatocyte Growth Factor (HGF)-mediated Lamellipodia Formation, Reactive Oxygen Species (ROS) Generation, and Motility of Lung Endothelial Cells

Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.

Role of reactive oxygen species in the regulation of cardiac contractility

Increased production of reactive oxygen species (ROS) has been linked to the pathogenesis of contractile dysfunction in heart failure. However, it is unclear whether ROS can regulate physiological cellular processes in the myocardium. Here, we characterized the role of endogenous ROS production in the acute regulation of cardiac contractility in the intact rat heart. In isolated perfused rat hearts, endothelin-1 (ET-1, 1 nmol/L) stimulated ROS formation in the left ventricle, which was prevented by the antioxidant N-acetylcysteine and the NAD(P)H oxidase inhibitor apocynin. N-acetylcysteine, the superoxide dismutase mimetic MnTMPyP, and apocynin significantly attenuated ET-1-mediated inotropic effect, which was accompanied by inhibition of extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Moreover, the mitochondrial K ATP channel blocker 5-HD, and the mitochondrial large conductance calcium activated potassium channel blocker paxilline, but not the sarcolemmal K ATP channel blocker HMR 1098 attenuated the inotropic response to ET-1. However, ET-1-induced ROS generation was not abolished by inhibiting mitochondrial K ATP channel opening. In contrast to ET-1 stimulation, the positive inotropic effect of β 1-adrenergic receptor agonist dobutamine (250 nmol/L) was significantly augmented by N-acetylcysteine and apocynin. Moreover, dobutamine-induced phospholamban phosphorylation was markedly enhanced by apocynin. In conclusion, NAD(P)H oxidase-derived ROS play a physiological role in the acute regulation of cardiac contractility in the intact rat heart. Our results reveal that ET-1-induced increase in cardiac contractility is partially dependent on enhanced ROS generation, which in turn, activates the ERK1/2 pathway. On the other hand, β-adrenergic receptor-induced positive inotropic effect and phospholamban phosphorylation is enhanced by NAD(P)H oxidase inhibition.

The effects of estrogen receptors α‐ and β‐specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.

Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and β-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from β-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

Aβ Oligomers Induce Neuronal Oxidative Stress through an N-Methyl-D-aspartate Receptor-dependent Mechanism That Is Blocked by the Alzheimer Drug Memantine

Oxidative stress is a major aspect of Alzheimer disease (AD) pathology. We have investigated the relationship between oxidative stress and neuronal binding of Abeta oligomers (also known as ADDLs). ADDLs are known to accumulate in brain tissue of AD patients and are considered centrally related to pathogenesis. Using hippocampal neuronal cultures, we found that ADDLs stimulated excessive formation of reactive oxygen species (ROS) through a mechanism requiring N-methyl-d-aspartate receptor (NMDA-R) activation. ADDL binding to neurons was reduced and ROS formation was completely blocked by an antibody to the extracellular domain of the NR1 subunit of NMDA-Rs. In harmony with a steric inhibition of ADDL binding by NR1 antibodies, ADDLs that were bound to detergent-extracted synaptosomal membranes co-immunoprecipitated with NMDA-R subunits. The NR1 antibody did not affect ROS formation induced by NMDA, showing that NMDA-Rs themselves remained functional. Memantine, an open channel NMDA-R antagonist prescribed as a memory-preserving drug for AD patients, completely protected against ADDL-induced ROS formation, as did other NMDA-R antagonists. Memantine and the anti-NR1 antibody also attenuated a rapid ADDL-induced increase in intraneuronal calcium, which was essential for stimulated ROS formation. These results show that ADDLs bind to or in close proximity to NMDA-Rs, triggering neuronal damage through NMDA-R-dependent calcium flux. This response provides a pathologically specific mechanism for the therapeutic action of memantine, indicates a role for ROS dysregulation in ADDL-induced cognitive impairment, and supports the unifying hypothesis that ADDLs play a central role in AD pathogenesis.

Cell signalling by oxidized lipids and the role of reactive oxygen species in the endothelium

The controlled formation of ROS (reactive oxygen species) and RNS (reactive nitrogen species) is now known to be critical in cellular redox signalling. As with the more familiar phosphorylation-dependent signal transduction pathways, control of protein function is mediated by the post-translational modification at specific amino acid residues, notably thiols. Two important classes of oxidant-derived signalling molecules are the lipid oxidation products, including those with electrophilic reactive centres, and decomposition products such as lysoPC (lysophosphatidylcholine). The mechanisms can be direct in the case of electrophiles, as they can modify signalling proteins by post-translational modification of thiols. In the case of lysoPC, it appears that secondary generation of ROS/RNS, dependent on intracellular calcium fluxes, can cause the secondary induction of H2O2 in the cell. In either case, the intracellular source of ROS/RNS has not been defined. In this respect, the mitochondrion is particularly interesting since it is now becoming apparent that the formation of superoxide from the respiratory chain can play an important role in cell signalling, and oxidized lipids can stimulate ROS formation from an undefined source. In this short overview, we describe recent experiments that suggest that the cell signalling mediated by lipid oxidation products involves their interaction with mitochondria. The implications of these results for our understanding of adaptation and the response to stress in cardiovascular disease are discussed.

Cell signalling by oxidized lipids and the role of reactive oxygen species in the endothelium.

The controlled formation of ROS (reactive oxygen species) and RNS (reactive nitrogen species) is now known to be critical in cellular redox signalling. As with the more familiar phosphorylation-dependent signal transduction pathways, control of protein function is mediated by the post-translational modification at specific amino acid residues, notably thiols. Two important classes of oxidant-derived signalling molecules are the lipid oxidation products, including those with electrophilic reactive centres, and decomposition products such as lysoPC (lysophosphatidylcholine). The mechanisms can be direct in the case of electrophiles, as they can modify signalling proteins by post-translational modification of thiols. In the case of lysoPC, it appears that secondary generation of ROS/RNS, dependent on intracellular calcium fluxes, can cause the secondary induction of H2O2 in the cell. In either case, the intracellular source of ROS/RNS has not been defined. In this respect, the mitochondrion is particularly interesting since it is now becoming apparent that the formation of superoxide from the respiratory chain can play an important role in cell signalling, and oxidized lipids can stimulate ROS formation from an undefined source. In this short overview, we describe recent experiments that suggest that the cell signalling mediated by lipid oxidation products involves their interaction with mitochondria. The implications of these results for our understanding of adaptation and the response to stress in cardiovascular disease are discussed.

Development and Application of an Electron Spin Resonance Spectrometry Method for the Determination of Oxygen Free Radical Formation by Particulate Matter

Exposure to increased levels of ambient particulate matter (PM) are associated with several health effects, including cardiopulmonary diseases. The formation of reactive oxygen species (ROS) is thought to play an important role in the induction of these health effects. To quantify the ROS generating capacityof PM,we developed an improved electron spin resonance (ESR) spectrometry-based method. ROS formation was measured directly on PM-containing filters, thereby avoiding the selective extraction of components and loss of material or reactivity, which is likely to occur during filter extraction. Also, ascorbic acid was added to stimulate ROS formation. This method was applied to PM10 samples originating from different sources. The radical generating capacity of PM10 from both gasoline and diesel engine exhaust was significantly higher as compared to that of PM10 from ambient or indoor air. Furthermore, in urban PM10 and PM2.5, ROS-generating capacity significantly correlated with concentrations of polycyclic aromatic hydrocarbon content and particular transition metals. This indicates thatthis improved ESR method may be a valuable tool for evaluating the relationship between ROS formation by PM and the adverse health effects associated with this type of air pollution.

The Role of Reactive Oxygen Species in γ-Globin Gene Activation by Fetal Hemoglobin Inducers.

Enhancing γ-globin expression is an effective therapeutic strategy for sickle cell disease. Fetal hemoglobin (HbF) inducers such as sodium butyrate (NaB), trichostatin A (TSA) and hydroxyurea (HU) have been studied extensively to ascertain molecular and cell signaling mechanisms for γ-gene activation. Reactive oxygen species (ROS) including nitric oxide, superoxide, hydroxyl radicals and hydrogen peroxide stimulate signaling through the cyclic guanosine monophosphate (cGMP) and p38 MAPK pathways. Indirect evidence supports HU as a nitric oxide donor thus providing a mechanism for HbF induction via cGMP activation. Recently, we and others demonstrated a role for p38 signaling in γ-gene induction by HU as well as NaB, TSA and apicidin. However, the ability of HbF inducers to stimulate ROS formation as a mechanism for γ-globin activation via cGMP or p38 MAPK signaling has not been determined. To clarify this mechanism we measured ROS levels using 2′,7′-dichlorofluorescin diacetate which emits fluorescence at a 529 nm wavelength when activated by hydrogen peroxide. We correlated ROS levels with p38 phosphorylation and γ-globin mRNA levels measured by western blot and real-time PCR respectively. Studies were also completed in the absence and presence of myxothiazol, a known inhibitor of ROS formation to confirm our findings. The positive controls menadione and hemin increased ROS levels 4-fold and 3-fold with a concomitant 6.5-fold and 4-fold increase in γ-globin mRNA respectively. Pretreatment with myxothiazol inhibited maximal HbF induction by both agents. By contrast, NaB and TSA did not increase ROS levels in K562 cells therefore they activate γ-globin by a ROS-independent, p38 MAPK-dependent mechanism. We concluded that ROS play a major role in the mechanism for HbF induction by hemin and the vitamin K derivative, menadione. Investigations were completed which confirmed the ability of HU to stimulate ROS formation as well. Studies are underway to determine the role of hydrogen peroxide and/or nitric oxide in HbF induction by HU via cGMP/p38 MAPK cell signaling.

Antagonistic Cross-talk between Rac and Cdc42 GTPases Regulates Generation of Reactive Oxygen Species

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b(558) and p67(phox) oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b(558) that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b(558) in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.

Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells.

ANG II induces protein synthesis through the serine-threonine kinase Akt/protein kinase B (PKB) in mesangial cells (MCs). The mechanism(s) of activation of Akt/PKB particularly by G protein-coupled receptors, however, is not well characterized. We explored the role of the small GTPase Rac1, a component of the phagocyte NADPH oxidase, and the gp91phox homologue Nox4/Renox in this signaling pathway. ANG II causes rapid activation of Rac1, an effect abrogated by phospholipase A2 inhibition and mimicked by arachidonic acid (AA). Northern blot analysis revealed high levels of Nox4 transcript in MCs and transfection with antisense (AS) oligonucleotides for Nox4 markedly decreased NADPH-dependent reactive oxygen species (ROS)-producing activity. Dominant negative Rac1 (N17Rac1) as well as AS Nox4 inhibited ROS generation in response to ANG II and AA, whereas constitutively active Rac1 stimulated ROS formation. Moreover, N17Rac1 blocked stimulation of NADPH oxidase activity by AA. N17Rac1 or AS Nox4 abolished ANG II- or AA-induced activation of the hypertrophic kinase Akt/PKB. In addition, AS Nox4 inhibited ANG II-induced protein synthesis. These data provide the first evidence that activation by AA of a Rac1-regulated, Nox4-based NAD(P)H oxidase and subsequent generation of ROS mediate the effect of ANG II on Akt/PKB activation and protein synthesis in MCs.

Endothelial NADPH oxidase: mechanism of activation by low-density lipoprotein.

Exposure to atherogenic levels of low-density lipoprotein (LDL) causes elevated reactive oxygen species (ROS) production by human endothelial cells (ECs). NADPH oxidase is thought to be the main source of ROS generated by LDL-activated ECs. The mechanism by which this lipoprotein activates endothelial NADPH oxidase is incompletely understood. To gain further insight into the signaling pathway, the authors have examined the effects of inhibitors to various signal transducing enzymes, including the G(i)-protein coupled receptor (pertussis toxin), Src tyrosine kinase (PP1), phospholipase C-gamma (U73122), phosphatidylinositol 3-kinase (LY294002), p42/p44 mitogen-activated protein kinase (MAPK) kinase (PD98059), p38 MAPK (SB203580), protein kinase C (Ro 318220, GF 109203X, Go 6976), and cytosolic phospholipase A(2) (AACOCF3), on the ROS-producing capacity ECs activated by LDL. Exposure of cultured ECs to LDL (0.45 mg protein/mL) stimulated ROS formation, as measured using a 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate assay. This effect was partially inhibited by Ro 318220, GF 109203X, U73122, and SB203580, and blocked or nearly completely inhibited by PP1, pertussis toxin, LY294002, PD98059, and AACOCF3. Only a partial, minor inhibition occurred with the protein kinase C inhibitor, Go 6976. These results are most consistent with LDL activating endothelial NADPH oxidase, predominantly through a signaling pathway that leads to cytosolic phospholipase A(2) activation.

Native LDL induces proliferation of human vascular smooth muscle cells via redox-mediated activation of ERK 1/2 mitogen-activated protein kinases.

This study investigated mechanisms underlying native low-density lipoprotein (LDL)-stimulated proliferation of human vascular smooth muscle cells (VSMC). Experiments were performed to determine whether native LDL affects reactive oxygen species (ROS) formation and activity of extracellular signal-regulated kinase 1/2 (ERK1/2), and whether redox-sensitive pathways contribute to LDL-induced cell proliferation. Native LDL (100 microg/mL, 24 hours) increased cell proliferation (to 303 to 388% of control, P<0.0001) as determined by [methyl-(3)H] thymidine incorporation. This effect was completely blocked either by the antioxidants N-acetylcysteine, Tiron, or nordihydroguaiaretic acid; the flavin-inhibitor diphenylene iodonium; or superoxide dismutase (all P<0.0001), and partly blocked by ERK-inhibitor PD98059 or meclofenamate (P<0.01). Exposure of VSMC to native LDL for 20 minutes stimulated ROS formation, measured by dichlorodihydrofluorescein oxidation, and increased ERK1/2 activity by 3.1-fold (P<0.001). The latter effect was sensitive to MEK1/2 inhibitor PD98059 and Tiron (P<0.001), and in part to N-acetylcysteine or diphenylene iodonium (P<0.05). These results demonstrate that native LDL induces acute formation of ROS and subsequent activation of redox-sensitive ERK 1/2 mitogen-activated protein kinases, pathways that appear to be important for mitogenic signaling of native LDL in human vascular smooth muscle cells.

Oxidative inactivation of cytochrome P-450 1A (CYP1A) stimulated by 3,3',4,4'-tetrachlorobiphenyl: production of reactive oxygen by vertebrate CYP1As.

Microsomal cytochrome P-450 1A (CYP1A) in a vertebrate model (the teleost fish scup) is inactivated by the aryl hydrocarbon receptor agonist 3,3',4,4'-tetrachlorobiphenyl (TCB). Here, the mechanism of CYP1A inactivation and its relationship to reactive oxygen species (ROS) formation were examined by using liver microsomes from scup and rat and expressed human CYP1As. In vitro inactivation of scup CYP1A activity 7-ethoxyresorufin O-deethylation by TCB was time dependent, NADPH dependent, oxygen dependent, and irreversible. TCB increased microsomal NADPH oxidation rates, and CYP1A inactivation was lessened by adding cytochrome c. CYP1A inactivation was accompanied by loss of spectral P-450, a variable loss of heme and a variable appearance of P-420. Rates of scup liver microsomal metabolism of TCB were < 0.5 pmol/min/mg, 25-fold less than the rate of P-450 loss. Non-heme iron chelators, antioxidant enzymes, and ROS scavengers had no influence on inactivation. Inactivation was accelerated by H(2)O(2) and azide but not by hydroxylamine or aminotriazole. TCB also inactivated rat liver microsomal CYP1A, apparently CYP1A1. Adding TCB to scup or rat liver microsomes containing induced levels of CYP1A, but not control microsomes, stimulated formation of ROS; formation rates correlated with native CYP1A1 content. TCB stimulated ROS formation by baculovirus-expressed human CYP1A1 but not CYP1A2. The results indicate that TCB uncouples the catalytic cycle of CYP1A, ostensibly CYP1A1, resulting in formation of ROS within the active site. These ROS may inactivate CYP1A or escape from the enzyme. ROS formed by CYP1A1 may contribute to the toxicity of planar halogenated aromatic hydrocarbons.

Reactive Oxygen Species Participate in Peroxynitrite-Induced Apoptosis in HL-60 Cells

Peroxynitrite (ONOO−) is a physiological product generated by the interaction of superoxide (O.−2) and nitric oxide (.NO). We have previously shown that peroxynitrite induces apoptosis in HL-60 cells. In the present study, we demonstrated that peroxynitrite generates reactive oxygen species (ROS) in HL-60 cells. Brief exposure of HL-60 cells to ONOO−induced elevation of lucigenin chemiluminescence, indicating generation of superoxide anion. Exogenous superoxide dismutase (SOD), a scavenger of O.−2, fully abolished the chemiluminescence response, further supporting this notion. Following O.−2generation, the accumulation of hydrogen peroxide (H2O2) was observed. The addition of SOD exacerbated but that of catalase attenuated peroxynitrite-induced DNA fragmentation, suggesting that this H2O2production contributes to the apoptotic process. In addition, pre-treatment of HL-60 cells with N-acetyl-L-cysteine (15 mM), a ROS scavenger, fully scavenged peroxynitrite-elicited ROS generation and effectively inhibited ONOO−-induced apoptosis, further enforcing this hypothesis. In summary, our results suggest that ONOO−-stimulated ROS formation may serve as a mechanism for the propagation of peroxynitrite-mediated apoptotic cell death in an intact cell system.

Contribution of hepatic cytochrome P450 systems to the generation of reactive oxygen species

The rate of generation of reactive oxygen species (ROS) in hepatic microsomes was assayed using a fluorescent probe. This rate was stimulated in a manner proportional to the concentration of NADPH present. NADH could not be substituted for NADPH, and an inhibitor of mixed-function oxidases (SKF 525A) blocked stimulation by NADPH. This suggested the involvement of cytochrome P450 oxidase systems in ROS formation. Low molecular weight iron salts may not have been involved in the stimulated ROS formation since deferoxamine failed to eliminate the oxidative response to NADPH. Catalase only partially inhibited, and glutathione peroxidase did not significantly inhibit this response, implying that hydrogen peroxide does not play a key role. However, since NADPH-enhanced generation of reactive oxygen species was totally prevented by Superoxide dismutase, Superoxide was an obligatory intermediate. The presence of toluene, ethanol or phenobarbital did not enhance the production of NADPH-effected reactive oxygen species; free radical production was maximal in the absence of substrates subject to oxidation by cytochrome P450 enzymes. Hepatic cytochrome P450 oxidases are likely to contribute significantly to overall ROS formation, even under basal conditions where mixed-function oxidases are not induced.