Tert-butyl Hydroperoxide Exposure Research Articles

Modulating glutathione thiol status alters pancreatic β-cell morphogenesis in the developing zebrafish (Danio rerio) embryo

Emerging evidence suggests that redox-active chemicals perturb pancreatic islet development. To better understand potential mechanisms for this, we used zebrafish (Danio rerio) embryos to investigate roles of glutathione (GSH; predominant cellular redox buffer) and the transcription factor Nrf2a (Nfe2l2a; zebrafish Nrf2 co-ortholog) in islet morphogenesis. We delineated critical windows of susceptibility to redox disruption of β-cell morphogenesis, interrogating embryos at 24, 48 and 72 h post fertilization (hpf) and visualized Nrf2a expression in the pancreas using whole-mount immunohistochemistry at 96 hpf. Chemical GSH modulation at 48 hpf induced significant islet morphology changes at 96 hpf. Pro-oxidant exposures to tert-butylhydroperoxide (77.6 μM; 10-min at 48 hpf) or tert-butylhydroquinone (1 μM; 48-56 hpf) decreased β-cell cluster area at 96 hpf. Conversely, exposures to antioxidant N-acetylcysteine (bolsters GSH pools; 100 μM; 48-72 hpf) or sulforaphane (activates Nrf2a; 20 μM; 48-72 hpf) significantly increased islet areas. Nrf2a was also stabilized in β-cells: 10-min exposures to 77.6 μM tert-butylhydroperoxide significantly increased Nrf2a protein compared to control islet cells that largely lack stabilized Nrf2a; 10-min exposures to higher (776 μM) tert-butylhydroperoxide concentration stabilized Nrf2a throughout the pancreas. Using biotinylated-GSH to visualize in situ protein glutathionylation, islet cells displayed high protein glutathionylation, indicating oxidized GSH pools. The 10-min high (776 μM) tert-butylhydroperoxide exposure (induced Nrf2a globally) decreased global protein glutathionylation at 96 hpf. Mutant fish expressing inactive Nrf2a were protected against tert-butylhydroperoxide-induced abnormal islet morphology. Our data indicate that disrupted redox homeostasis and Nrf2a stabilization during pancreatic β-cell development impact morphogenesis, with implications for disease states at later life stages. Our work identifies a potential molecular target (Nrf2) that mediates abnormal β-cell morphology in response to redox disruptions. Moreover, our findings imply that developmental exposure to exogenous stressors at distinct windows of susceptibility could diminish the reserve redox capacity of β-cells, rendering them vulnerable to later-life stresses and disease.

Crocetin Prevents RPE Cells from Oxidative Stress through Protection of Cellular Metabolic Function and Activation of ERK1/2.

Age-related macular degeneration (AMD) is a leading cause for visual impairment in aging populations with limited established therapeutic interventions available. Oxidative stress plays an essential role in the pathogenesis of AMD, damaging the retinal pigment epithelium (RPE), which is essential for the function and maintenance of the light-sensing photoreceptors. This study aimed to evaluate the effects of crocetin, one of the main components of Saffron, on an in vitro RPE model of tert-butyl hydroperoxide (TBHP) induced oxidative stress using ARPE19 cells. The effects of crocetin were assessed using lactate de-hydrogenase (LDH) and ATP assays, as well as immunocytochemistry for cell morphology, junctional integrity, and nuclear morphology. The mechanism of crocetin action was determined via assessment of energy production pathways, including mitochondrial respiration and glycolysis in real-time as well as investigation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and distribution. Our results show that crocetin pre-treatment protects ARPE19 cells from TBHP-induced LDH release, intracellular ATP depletion, nuclear condensation, and disturbance of junctional integrity and cytoskeleton. The protective effect of crocetin is mediated via the preservation of energy production pathways and activation of ERK1/2 in the first minutes of TBHP exposure to potentiate survival pathways. The combined data suggest that a natural antioxidant, such as crocetin, represents a promising candidate to prevent oxidative stress in RPE cells and might halt or delay disease progression in AMD.

Combining antioxidant astaxantin and cholinesterase inhibitor huperzine A boosts neuroprotection.

Oxidative stress is a pathophysiological condition resulting in neurotoxicity, which is possibly associated with neurodegenerative disorders. In this study, the antioxidative effects of the antioxidant astaxanthin (AXT) in combination with huperzine A (HupA), which is used as a cholinesterase inhibitor for the treatment of Alzheimer's disease, were investigated. PC12 cells were treated with either tert-butyl hydroperoxide (TBHP), or with the toxic version of β-amyloid, Aβ25–35, to induce oxidative stress and neurotoxicity. Cell viability, morphology, lactate dehydrogenase (LDH) release, intracellular accumulation of reactive oxygen species (ROS), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were determined, while neuroprotection was also monitored using an MTT assay. It was found that combining AXT with HupA significantly increased the viability of PC12 cells, prevented membrane damage (as measured by LDH release), attenuated intracellular ROS formation, increased SOD activity and decreased the level of MDA after TBHP exposure when compared to these drugs administered alone. Pretreatment with HupA and AXT decreased toxic damage produced by Aβ25–35. These data indicated that combining an antioxidant with a cholinesterase inhibitor increases the degree of neuroprotection; with future investigation this could be a potential therapy used to decrease neurotoxicity in the brain.

Protein kinase Cε targets respiratory chain and mitochondrial membrane potential but not F0 F1 -ATPase in renal cells injured by oxidant.

We have previously shown that protein kinase Cε (PKCε) is involved in mitochondrial dysfunction in renal proximal tubular cells (RPTC). This study examined mitochondrial targets of active PKCε in RPTC injured by the model oxidant tert-butyl hydroperoxide (TBHP). TBHP exposure augmented the levels of phosphorylated (active) PKCε in mitochondria, which suggested translocation of PKCε to mitochondria after oxidant exposure. Oxidant injury decreased state 3 respiration, adenosine triphosphate (ATP) production, ATP content, and complex I activity. Further, TBHP exposure increased ΔΨm and production of reactive oxygen species (ROS), and induced mitochondrial fragmentation and RPTC death. PKCε activation by overexpressing constitutively active PKCε exacerbated decreases in state 3 respiration, complex I activity, ATP content, and augmented RPTC death. In contrast, inhibition of PKCε by overexpressing dnPKCε mutant restored state 3 respiration, respiratory control ratio, complex I activity, ΔΨm , and ATP production and content, but did not prevent decreases in F0 F1 -ATPase activity. Inhibition of PKCε prevented oxidant-induced production of ROS and mitochondrial fragmentation, and reduced RPTC death. We conclude that activation of PKCε mediates: (a) oxidant-induced changes in ΔΨm , decreases in mitochondrial respiration, complex I activity, and ATP content; (b) mitochondrial fragmentation; and (c) RPTC death. In contrast, oxidant-induced inhibition of F0 F1 -ATPase activity is not mediated by PKCε. These results show that, in contrast to the protective effects of PKCε in the heart, PKCε activation is detrimental to mitochondrial function and viability in RPTC and mediates oxidant-induced injury.

ERK1/2 Activation Following Renal Injury Is Required For Kidney Injury Molecule‐1 Production

BackgroundBecause substantial damage can occur before detection of acute kidney injury (AKI) new early predictor biomarkers are needed, such as kidney injury molecule‐1 (KIM‐1). KIM‐1 is a transmembrane glycoprotein that is expressed at very low levels in healthy kidney and is highly upregulated during injury. KIM‐1 is shed during injury and can be detected in blood and urine. While KIM‐1 has been studied in various models of AKI, the regulatory mechanisms leading to KIM‐1 upregulation remains limited. Here, we explored the role of ERK1/2 activation on KIM‐1 expression following toxicant exposure to proximal tubule cells and renal ischemia reperfusion injury (IRI) and LPS‐induced sepsis in mice.MethodsMouse kidney proximal tubule cells (TK) were treated with varying concentrations of hydroxyurea (HU), hydrogen peroxide (H2O2), and tert‐butyl‐hydroperoxide (TBHP) with and without pretreatment with the MEK1/2 inhibitor trametinib (10 nM). Trametinib (1mg/kg) was administered 1 hr before renal IRI to C57Bl/6 mice and were euthanized at 3 hr and 24 hr. TLR4 KO and WT mice underwent 3 hr renal IRI or septic AKI for 18 hr using LPS (10 mg/kg) injection. Signaling pathways were explored using RT‐qPCR and immunoblot analysis and kidney function was measured using BUN and serum creatinine.ResultsHU, H2O2, and TBHP exposure to TK cells for 24 hr caused ERK1/2 phosphorylation and upregulated KIM‐1 mRNA. Pretreatment with trametinib prevented KIM‐1 up‐regulation and ERK1/2 phosphorylation at 24 hr. At 3 hr post renal IRI KIM‐1 mRNA increased 4‐fold while pretreatment with trametinib resulted in a 2‐fold increase in KIM‐1 mRNA. Serum creatinine increased 4‐fold 3 hr after IRI and was attenuated by trametinib pretreatment. Cortical KIM‐1 mRNA increased 600‐fold 24 hr after IRI and was reduced 66% using trametinib in IRI. KIM‐1 protein was observed at 24 hr but not at 3 hr post IRI. Trametinib decreased KIM‐1 protein in the kidney 24 hr post IRI. TLR4 KO mice subjected to IRI exhibited increased KIM‐1 mRNA and ERK1/2 phosphorylation after 3 hr. In contrast, TLR4 KO mice exposed to LPS did not exhibit increased KIM‐1 mRNA or protein, nor activated ERK1/2 after 18 hr. WT mice treated with LPS had increased KIM‐1 mRNA and protein and ERK1/2 phosphorylation.ConclusionWe have linked pathophysiological ERK1/2 phosphorylation to the regulation of KIM‐1 within the kidney cortex. ERK1/2 phosphorylation and KIM‐1 mRNA increased following toxicant exposure in TK cells, IRI in mice, and LPS in mice, and trametinib attenuated these increases. LPS‐induced AKI required TLR4 for ERK1/2 activation and subsequent upregulation of KIM‐1. In contrast, IRI mice did not require TLR4 for ERK1/2 activation. These results demonstrate that ERK1/2 is a key initiator of renal injury.Support or Funding InformationGM084147 ‐ RGSF31DK105782 ‐ JBC

Protective effects of unsaponifiable matter from rice bran on oxidative damage by modulating antioxidant enzyme activities in HepG2 cells

Rice bran is a byproduct of the rice milling process. It contains high levels of bioactive phytochemicals such as phytosterols, tocopherols, tocotrienols, γ-oryzanol, triterpene alcohol, and other minor compounds, which have antioxidant and health-promoting properties. The objective of this study was to evaluate the protective effect of unsaponifiable matter (USM) from rice bran against oxidative damages induced by tert-butyl hydroperoxide (TBHP) in HepG2 cells. Levels of cellular reactive oxygen species (ROS), cellular lipid peroxidation, reduced glutathione (GSH), and antioxidant enzyme activity were evaluated as biomarkers of the cellular oxidative status. HepG2 cells were pretreated with various concentrations of USM (0–100 μg/mL) for 12 h prior to TBHP exposure. Pretreatment of HepG2 cells with USM prevented the cell damage induced by TBHP in a dose-dependent manner. Cellular generation of ROS, formation of malondialdehyde (MDA), and depletion of GSH induced by TBHP were prevented by pretreatment with USM. In addition, treatment with TBHP increased the activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. However, pretreatment with USM significantly reduced the activities of these enzymes. These results indicate that treatment of HepG2 cells with USM confers significant protection against oxidative damage by modulating ROS production, GSH levels, and antioxidant enzyme activity.

Elucidating the response of Kluyveromyces lactis to arsenite and peroxide stress and the role of the transcription factor KlYap8

All organisms need to sense and respond to a range of stress conditions. In this study, we used transcriptional profiling to identify genes and cellular processes that are responsive during arsenite and tert-butyl hydroperoxide exposure in Kluyveromyces lactis. Many arsenite-responsive genes encode proteins involved in redox processes, protein folding and stabilization, and transmembrane transport. The majority of peroxide-responsive genes encode functions related to transcription, translation, redox processes, metabolism and transport. A substantial number of these stress-regulated genes contain binding motifs for the AP-1 like transcription factors KlYap1 and KlYap8. We demonstrate that KlYap8 binds to and regulates gene expression through a 13 base-pair promoter motif, and that KlYap8 provides protection against arsenite, antimonite, cadmium and peroxide toxicity. Direct transport assays show that Klyap8Δ cells accumulate more arsenic and cadmium than wild type cells and that the Klyap8Δ mutant is defective in arsenic and cadmium export. KlYap8 regulates gene expression in response to both arsenite and peroxide, and might cooperate with KlYap1 in regulation of specific gene targets. Comparison of KlYap8 with its Saccharomyces cerevisiae orthologue ScYap8 indicates that KlYap8 senses and responds to multiple stress signals whereas ScYap8 is only involved in the response to arsenite and antimonite. Thus, our data suggest that functional specialization of ScYap8 has occurred after the whole genome duplication event. This is the first genome-wide stress response analysis in K. lactis and the first demonstration of KlYap8 function.

Resistance to oxidative stress is not associated with the exceptional longevity of the freshwater pearl mussel, Margaritifera margaritifera nor three unionid species

Bivalve molluscs are newly discovered models of successful ageing. Here, we test the hypothesis that extreme longevity of freshwater mussels is associated with an enhanced resistance to oxidative stress. We assess whether resistance to oxidative stress might be causally involved in the exceptional longevity exhibited by the freshwater pearl mussel Margaritifera margaritifera. We compared resistance to oxidative stress and total haemocyte counts, a health status biomarker in M. margaritifera (maximum lifespan potential 190 years) with three other freshwater bivalve species spanning a range of longevities. Previous studies of the comparative stress resistance and longevity of marine bivalves provide evidence for the hypothesis that an association exists between longevity and not only an enhanced resistance to oxidative stress but also a general resistance to multiplex stressors. We compared baseline total haemocyte counts, age-related changes, and responses to exposure to the oxidative stressor tert-butyl hydroperoxide (TBHP). Surprisingly our data does not support the premise that extreme longevity in M. margaritifera is associated with enhanced resistance to oxidative stress. In comparison with its shorter-lived counter parts M. margaritifera was the least resistance to oxidative stress. Following TBHP exposure, no association between longevity and resistance to oxidative stress-induced mortality nor a marked resistance to oxidative stress-induced declines in total haemocyte counts were observed. The results suggest longevity evolved separately in freshwater mussels and this group warrants further attention from biogerontologists because such study may provide novel insights not detected through the study of the marine members of the class, where most attention is currently focused.

Protein kinase C-α interaction with iHSP70 in mitochondria promotes recovery of mitochondrial function after injury in renal proximal tubular cells

This study determined the role of PKC-α and associated inducible heat shock protein 70 (iHSP70) in the repair of mitochondrial function in renal proximal tubular cells (RPTCs) after oxidant injury. Wild-type PKC-α (wtPKC-α) and an inactive PKC-α [dominant negative dn; PKC-α] mutant were overexpressed in primary cultures of RPTCs, and iHSP70 levels and RPTC regeneration were assessed after treatment with the oxidant tert-butylhydroperoxide (TBHP). TBHP exposure increased ROS production and induced RPTC death, which was prevented by ferrostatin and necrostatin-1 but not by cyclosporin A. Overexpression of wtPKC-α maintained mitochondrial levels of active PKC-α, reduced cell death, and accelerated proliferation without altering ROS production in TBHP-injured RPTCs. In contrast, dnPKC-α blocked proliferation and monolayer regeneration. Coimmunoprecipitation and proteomic analysis demonstrated an association between inactive, but not active, PKC-α and iHSP70 in mitochondria. Mitochondrial iHSP70 levels increased as levels of active PKC-α decreased after injury. Overexpression of dnPKC-α augmented, whereas overexpression of wtPKC-α abrogated, oxidant-induced increases in mitochondrial iHSP70 levels. iHSP70 overexpression (1) maintained mitochondrial levels of phosphorylated PKC-α, (2) improved the recovery of state 3 respiration and ATP content, (3) decreased RPTC death (an effect abrogated by cyclosporine A), and (4) accelerated proliferation after oxidant injury. In contrast, iHSP70 inhibition blocked the recovery of ATP content and exacerbated RPTC death. Inhibition of PKC-α in RPTC overexpressing iHSP70 blocked the protective effects of iHSP70. We conclude that active PKC-α maintains mitochondrial function and decreases cell death after oxidant injury. iHSP70 is recruited to mitochondria in response to PKC-α dephosphorylation and associates with and reactivates inactive PKC-α, which promotes the recovery of mitochondrial function, decreases RPTC death, and improves regeneration.

The Role of Endoplasmic Reticulum Ca2+-Independent Phospholipase A2γ in Oxidant-Induced Lipid Peroxidation, Ca2+ Release, and Renal Cell Death

Oxidant-induced lipid peroxidation and cell death are major components of ischemia/reperfusion and toxicant injury. Our previous studies showed that renal proximal tubular cells (RPTCs) express Ca(2+)-independent phospholipase A(2)γ (iPLA(2)γ) in endoplasmic reticulum (ER) and mitochondria and that iPLA(2)γ is cytoprotective. Our present studies reveal the role of ER-iPLA(2)γ in oxidant-induced ER lipid peroxidation, Ca(2+) release, and cell death. Oxidant tert-butyl hydroperoxide (TBHP) caused ER lipid peroxidation and Ca(2+) release in isolated rabbit kidney cortex microsomes. ER-iPLA(2)γ inhibition, using bromoenol lactone (BEL), potentiated both oxidant-induced ER lipid peroxidation and Ca(2+) release. Assessment of fatty acids using electrospray ionization-mass spectrometry revealed that ER-iPLA(2)γ mediates the TBHP-induced release of arachidonic acid (20:4), linoleic acid (18:2), and their oxidized forms (18:2-OH, 18:2-OOH, 20:4-OH, 20:4-OOH, 20:4-(OH)(3). iPLA(2)γ inhibition also accelerated oxidant-induced ER Ca(2+) release in RPTC. Depletion of ER Ca(2+) stores in RPTC with thapsigargin, an ER Ca(2+) pump inhibitor, prior to TBHP exposure reduced necrotic cell death and blocked the potentiation of TBHP-induced necrotic cell death by BEL. Together, these data provide strong evidence that ER-iPLA(2)γ protects renal cells from oxidant-induced necrotic cell death by releasing unsaturated and/or oxidized fatty acids from ER membranes, thereby preserving ER membrane integrity and preventing ER Ca(2+) release.

γ-Tocotrienol Protects against Mitochondrial Dysfunction and Renal Cell Death

Oxidative stress is a major mechanism of a variety of renal diseases. Tocopherols and tocotrienols are well known antioxidants. This study aimed to determine whether γ-tocotrienol (GT3) protects against mitochondrial dysfunction and renal proximal tubular cell (RPTC) injury caused by oxidants. Primary cultures of RPTCs were injured by using tert-butyl hydroperoxide (TBHP) in the absence and presence of GT3 or α-tocopherol (AT). Reactive oxygen species (ROS) production increased 300% in TBHP-injured RPTCs. State 3 respiration, oligomycin-sensitive respiration, and respiratory control ratio (RCR) decreased 50, 63, and 47%, respectively. The number of RPTCs with polarized mitochondria decreased 54%. F₀F₁-ATPase activity and ATP content decreased 31 and 65%, respectively. Cell lysis increased from 3% in controls to 26 and 52% at 4 and 24 h, respectively, after TBHP exposure. GT3 blocked ROS production, ameliorated decreases in state 3 and oligomycin-sensitive respirations and F₀F₁-ATPase activity, and maintained RCR and mitochondrial membrane potential (ΔΨ(m)) in injured RPTCs. GT3 maintained ATP content, blocked RPTC lysis at 4 h, and reduced it to 13% at 24 h after injury. Treatment with equivalent concentrations of AT did not block ROS production and cell lysis and moderately improved mitochondrial respiration and coupling. This is the first report demonstrating the protective effects of GT3 against RPTC injury by: 1) decreasing production of ROS, 2) improving mitochondrial respiration, coupling, ΔΨ(m), and F₀F₁-ATPase function, 3) maintaining ATP levels, and 4) preventing RPTC lysis. Our data suggest that GT3 is superior to AT in protecting RPTCs against oxidant injury and may prove therapeutically valuable for preventing renal injury associated with oxidative stress.

Extreme Longevity Is Associated With Increased Resistance to Oxidative Stress in Arctica islandica, the Longest-Living Non-Colonial Animal

We assess whether reactive oxygen species production and resistance to oxidative stress might be causally involved in the exceptional longevity exhibited by the ocean quahog Arctica islandica. We tested this hypothesis by comparing reactive oxygen species production, resistance to oxidative stress, antioxidant defenses, and protein damage elimination processes in long-lived A islandica with the shorter-lived hard clam, Mercenaria mercenaria. We compared baseline biochemical profiles, age-related changes, and responses to exposure to the oxidative stressor tert-butyl hydroperoxide (TBHP). Our data support the premise that extreme longevity in A islandica is associated with an attenuated cellular reactive oxygen species production. The observation of reduced protein carbonyl concentration in A islandica gill tissue compared with M mercenaria suggests that reduced reactive oxygen species production in long-living bivalves is associated with lower levels of accumulated macromolecular damage, suggesting cellular redox homeostasis may determine life span. Resistance to aging at the organismal level is often reflected in resistance to oxidative stressors at the cellular level. Following TBHP exposure, we observed not only an association between longevity and resistance to oxidative stress-induced mortality but also marked resistance to oxidative stress-induced cell death in the longer-living bivalves. Contrary to some expectations from the oxidative stress hypothesis, we observed that A islandica exhibited neither greater antioxidant capacities nor specific activities than in M mercenaria nor a more pronounced homeostatic antioxidant response following TBHP exposure. The study also failed to provide support for the exceptional longevity of A islandica being associated with enhanced protein recycling. Our findings demonstrate an association between longevity and resistance to oxidative stress-induced cell death in A islandica, consistent with the oxidative stress hypothesis of aging and provide justification for detailed evaluation of pathways involving repair of free radical-mediated macromolecular damage and regulation of apoptosis in the world's longest-living non-colonial animal.

Signaling of Mitochondrial Biogenesis following Oxidant Injury

Mitochondrial dysfunction is a common consequence of ischemia-reperfusion and drug injuries. For example, sublethal injury of renal proximal tubular cells (RPTCs) with the model oxidant tert-butylhydroperoxide (TBHP) causes mitochondrial injury that recovers over the course of six days. Although regeneration of mitochondrial function is integral to cell repair and function, the signaling pathway of mitochondrial biogenesis following oxidant injury has not been examined. A 10-fold overexpression of the mitochondrial biogenesis regulator PPAR-gamma cofactor-1alpha (PGC-1alpha) in control RPTCs resulted in a 52% increase in mitochondrial number, a 27% increase in respiratory capacity, and a 30% increase in mitochondrial protein markers, demonstrating that PGC-1alpha mediates mitochondrial biogenesis in RPTCs. RPTCs sublethally injured with TBHP exhibited a 50% decrease in mitochondrial function and increased mitochondrial autophagy. Compared with the controls, PGC-1alpha levels increased 12-fold on days 1, 2, and 3 post-injury and returned to base line on day 4 as mitochondrial function returned. Inhibition p38 MAPK blocked the up-regulation of PGC-1alpha following oxidant injury, whereas inhibition of calcium-calmodulin-dependent protein kinase, calcineurin A, nitric-oxide synthase, and phosphoinositol 3-kinase had no effect. The epidermal growth factor receptor (EGFR) was activated following TBHP exposure, and the EGFR inhibitor AG1478 blocked the up-regulation of PGC-1alpha. Additional inhibitor studies revealed that the sequential activation of Src, p38 MAPK, EGFR, and p38 MAPK regulate the expression of PGC-1alpha following oxidant injury. In contrast, although Akt was activated following oxidant injury, it did not play a role in PGC-1alpha expression. We suggest that mitochondrial biogenesis following oxidant injury is mediated by p38 and EGFR activation of PGC-1alpha.

Inactivation of endoplasmic reticulum bound Ca2+-independent phospholipase A2 in renal cells during oxidative stress.

The purpose of this study was to determine the actions of oxidants on endoplasmic reticulum bound Ca(2+)-independent phospholipase A(2) (ER-iPLA(2)) and phospholipids in renal cells. Exposure of renal proximal tubule cells (RPTC) to the oxidants tert-butyl hydroperoxide (TBHP), cumene hydroperoxide, and cisplatin resulted in time- and concentration-dependent decreases in the activity of ER-iPLA(2). TBHP-induced ER-iPLA(2) inactivation was reversed by the addition of dithiothreitol to microsomes isolated from treated RPTC. TBHP also directly inactivated ER-iPLA(2) in microsomes isolated from untreated RPTC. Similar to RPTC, dithiothreitol prevented TBHP-induced ER-iPLA(2) inactivation in microsomes as did the reactive oxygen scavengers butylated hydroxytoluene and N,N'-diphenyl-p-phenylenediamine and the iron chelator deferoxamine. Electron paramagnetic resonance spin trapping demonstrated that TBHP initiated a carbon-centered radical after 1 min of exposure in microsomes, preceding ER-iPLA(2) inactivation, and further studies suggested that the formation of the carbon-centered radical species occurred after or in concert with the formation of oxygen-centered radicals. Phospholipid content was determined after TBHP exposure in the presence and absence of the ER-iPLA(2) inhibitor bromoenol lactone. Treatment of RPTC with TBHP resulted in 35% decreases in (16:0, 20:4)-phosphatidylethanolamine (PtdEtn), (18:0, 18:1)-plasmenylethanolamine (PlsEtn), a 30% decrease in (16:0, 18:3)-phosphatidylcholine (PtdCho), and a 25% decrease in (16:0, 20:4)-phosphatidylcholine (PtdCho). In contrast, treatment of RPTC with bromoenol lactone before TBHP exposure decreased the content of 11 phospholipids, decreasing a majority of PlsEtn phospholipids 60%, and 4 of the 8 PlsCho phospholipids 40%, while PtdCho and PtdEtn were marginally affected compared with TBHP. These data demonstrate that ER-iPLA(2) is inactivated by oxidants, that the mechanism of inactivation involves the oxidation of ER-iPLA(2) sulfhydryl groups, and that ER-iPLA(2) inhibition increases oxidant-induced RPTC phospholipid loss.

Role of an endoplasmic reticulum Ca(2+)-independent phospholipase A(2) in oxidant-induced renal cell death.

Phospholipase A(2) (PLA(2)) hydrolyzes the sn-2 ester bond in phospholipids, releasing a fatty acid and a lysophospholipid. Recently, a novel 85-kDa membrane-bound-Ca(2+)-independent PLA(2) (iPLA(2)) was identified in insect and bacterial cells transfected with candidate PLA(2) sequences. However, few data exist demonstrating a membrane-bound-iPLA(2) in mammalian cells, its subcellular localization, or its physiological role. Herein, we demonstrate the expression of an 85-kDa endoplasmic reticulum (ER)-Ca(2+)-iPLA(2) (ER-iPLA(2)) in rabbit renal proximal tubule cells (RPTC) that is plasmalogen selective and is inhibited by the specific Ca(2+)-iPLA(2) inhibitor bromoenol lactone (BEL). RPTC exposed to tert-butylhydroperoxide for 24 h exhibited 20% oncosis compared with 2% in controls. Inhibition of ER-iPLA(2) with BEL before tert-butylhydroperoxide exposure resulted in 50% oncosis. To determine whether this effect was common to oxidants, we tested the ability of BEL to potentiate oncosis induced by cumene hydroperoxide, menadione, duraquinone, cisplatin, and the nonoxidant antimycin A. All oxidants tested produced oncosis after 24 h, and prior inhibition of ER-iPLA(2) potentiated oncosis at least twofold. In contrast, inhibition of ER-iPLA(2) did not alter antimycin A-induced oncosis. Lipid peroxidation increased from 1.4- to 5.2-fold in RPTC treated with BEL before oxidant exposure, whereas no change was seen in antimycin A-treated RPTC. These results are the first to demonstrate the expression and subcellular localization of an ER-iPLA(2). These results also suggest that ER-iPLA(2) functions to protect against oxidant-induced lipid peroxidation and oncosis.

Recovery of cellular functions following oxidant injury.

This study investigated the recovery of renal proximal tubule cellular (RPTC) functions following oxidant-induced sublethal injury. tert-Butylhydroperoxide (TBHP) treatment resulted in 24% cell death and loss 4 h following the exposure. The remaining sublethally injured RPTC proliferated, and monolayer DNA content returned to control values on day 4 following TBHP exposure. Basal oxygen consumption (Qo2) and ATP content in sublethally injured RPTC were decreased 64 and 63%, respectively, at 4 h and returned to control values on day 6. Net lactate consumption decreased 71% at 4 h and returned to control values on day 4. In contrast, net glutamine consumption increased 2.7-fold at 4 h and returned to control values on day 6. Ouabain-sensitive Qo2, Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity, and Na(+)-coupled glucose transport were inhibited 77, 88, and 83%, respectively, at 4 h and recovered to control values on day 6. These data show that 1) mitochondrial function, Na(+)-K(+)-ATPase activity, active Na+ transport, and Na(+)-coupled glucose transport are decreased in sublethally injured RPTC following oxidant exposure and are repaired over time; 2) monolayer regeneration precedes the recovery of mitochondrial and transport functions, and 3) sublethal injury and subsequent regeneration are associated with alterations in metabolic substrate utilization. These results suggest that oxidant-induced sublethal injury to RPTC may contribute to renal dysfunction and that RPTC can repair and regain cellular functions following oxidant injury.

Regeneration of Renal Proximal Tubule Cells in Primary Culture Following Toxicant Injury: Response to Growth Factors

Growth factors may play an important role in the repair of the renal proximal tubule epithelium following toxic injury. This study investigated the regeneration of rabbit renal proximal tubule cell (RPTC) monolayers following exposure to the nephrotoxicants tert-butylhydroperoxide (TBHP) and 1,2-dichlorovinyl-L-cysteine (DCVC), and the effect of exogenous growth factors on the regeneration process. Confluent monolayers exposed to TBHP or DCVC for 1.5-2 hr were 23 and 43% confluent, respectively, after 24 hr. Confluency increased to 63 and 80% 4 days after TBHP or DCVC exposure, but decreased to 29 and 24% after 8 days. Monolayer DNA content did not increase after TBHP or DCVC exposure; however, monolayer protein/DNA ratio increased above control values after DCVC exposure. Recovery of confluency was not sensitive to RGD-containing peptides that inhibit the binding of integrins to extracellular matrix. Epidermal growth factor (EGF) treatment resulted in complete recovery of confluency, and protein and DNA contents 4-6 days after injury. Unlike EGF, IGF-1 or insulin treatment produced a small increase in confluency following TBHP exposure. These results suggest that hypertrophy following DCVC exposure and migration/spreading after TBHP and DCVC exposure play a partial and temporary role in the regeneration of RPTC monolayers, that in the absence of exogenous growth factors proliferation and complete regeneration of the monolayer does not occur, that toxicants may alter the production of mitogenic factors, and that EGF is a potent and efficacious growth factor in promoting regeneration.

Hydroperoxides selectively inhibit human erythrocyte membrane enzymes

Treatment of washed erythrocytes with tert-butyl hydroperoxide (0.5 m m, 10 min) inhibited basal Ca 2+ + Mg 2+-ATPase activity by 40% and calmodulin-stimulated activity by 54%. The inhibition was accompanied by the formation of methemoglobin and the aggregation of some membrane proteins into a high-molecular-weight polymer. Membranes, isolated from washed erythrocytes, showed a similar pattern of inhibition. Basal Ca 2+ + Mg 2+-ATPase activity was inhibited 50% at 10 min and 70% at 30 min while calmodulin-stimulated activity was inhibited 70% at 10 min and 84% at 30 min. Thiobarbituric acid-reactive products formed slowly during the first 10 min and then increased sharply between 10 and 30 min. The polymerization of membrane proteins was also observed during the tert-butyl hydroperoxide exposure. Inhibition of erythrocyte membrane enzymes was selective. The Na + + K +-stimulated Mg 2+ ATPase, like the Ca 2+ + Mg 2+-ATPase, was sensitive to membrane oxidation but the activities of Mg 2+-ATPase and acetylcholinesterase were less inhibited by tert-butyl hydroperoxide. Acetylcholinterase was found to be very resistant to hydroperoxide treatment with less than 10% loss of activity. The effects of two other hyproperoxides on enzyme inhibition were studied also. Cumene hydroperoxide (0.5 m m) was found to be as potent as tert-butyl hydroperoxide but hydrogen peroxide at 10 m m did not produce thiobarbituric acid-reactive products or inhibit Ca 2+ + Mg 2+-ATPase activity until after 20 min. The selective effects of peroxides on these enzyme activities are discussed.

Glutathione protects against exogenous oxidant injury to rabbit renal proximal tubules

Glutathione, comprising a major portion of cellular nonprotein thiols, plays a central role in a diverse group of cell metabolic functions. Glutathione and related enzyme systems have been shown to protect against both toxin and oxidant-induced injury in several organ systems. The role of glutathione in protecting renal epithelia against oxidant stress has not been investigated previously. We report here the response of enriched, isolated rabbit renal proximal tubule segments to oxidant stress induced by tert-butyl hydroperoxide. In addition, the effects of glutathione depletion by various biochemical means and of exogenous glutathione supplementation on the response of tubule segments to tert-butyl hydroperoxide exposure are described. Depletion of cell glutathione by several distinct methods potentiates oxidant-induced injury. Augmentation of cellular glutathione affords significant protection against exogenous oxidant stress. The protective effect of glutathione may reside in its ability, in conjunction with glutathione peroxidase, to arrest the propagation of lipid peroxidation and, therefore, to minimize alterations in plasma membrane permeability. The results of this study do not exclude the possibility that glutathione prevents tert-butyl hydroperoxide induced oxidation of critical sulfhydryl groups of catalytic or structural proteins associated with control of cell cation homeostasis. These results confirm the important role of glutathione in protecting renal tubular epithelia against oxidant stress.