Accumulation of 15-deoxy-delta(12,14)-prostaglandin J2 adduct formation with Keap1 over time: effects on potency for intracellular antioxidant defence induction

The COX (cyclo-oxygenase) pathway generates the reactive lipid electrophile 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2), which forms covalent protein adducts that modulate cell signalling pathways. It has been shown that this regulates important biological responses, including protection against oxidative stress, and supports the proposal that 15d-PGJ2 has pharmacological potential. Protective pathways activated by 15d-PGJ2 include those controlling the synthesis of the intracellular antioxidants GSH and the enzyme HO-1 (haem oxygenase-1). The induction of the synthesis of these intracellular antioxidants is, in large part, regulated by covalent modification of Keap1 (Kelchlike erythroid cell-derived protein with 'capn'collar homologyassociated protein 1) by the lipid and the subsequent activation of the EpRE (electrophile-response element). For the first time, we show that the potency of 15d-PGJ2 as a signalling molecule in endothelial cells is significantly enhanced by the accumulation of the covalent adduct with 15d-PGJ2 and endogenous Keap1 over the time of exposure to the prostaglandin. The consequence of this finding is that signalling initiated by electrophilic lipids differs from agonists that do not form covalent adducts with proteins because the constant generation of very lowconcentrations of 15d-PGJ2 can lead to induction of GSH or HO-1. In the course of these studies we also found that a substantial amount (97-99%) of exogenously added 15d-PGJ2 is inactivated in the medium and does not enter the cells to initiate cell signalling. In summary, we propose that the accumulation of covalent adduct formation with signalling proteins provides a mechanism through which endogenous intracellular formation of electrophilic lipids from COX can exert an anti-inflammatory effect in vivo.     

Protective effect of rat aldo-keto reductase (AKR1C15) on endothelial cell damage elicited by 4-hydroxy-2-nonenal

4-Hydroxy-2-nonenal (HNE), a major reactive product of lipid peroxidation, is believed to play a central role in atherogenic actions triggered by oxidized lipoproteins. An aldo-keto reductase (AKR) 1C15 efficiently reduces HNE and is distributed in many rat tissues including endothelial cells. In this study, we investigated whether AKR1C15 acts as a protective factor against endothelial damage elicited by HNE and oxidized lipoproteins. Treatment of rat endothelial cells with HNE provoked apoptosis through reactive oxygen species (ROS) formation, mitochondrial dysfunction and caspase activation in the cells. AKR1C15 converted HNE into less toxic 1,4-dihydroxy-2-nonene, and its overexpression markedly decreased the susceptibility of the cells to HNE. The forced expression of AKR1C15 also significantly suppressed the loss of cell viability caused by oxidized low-density lipoprotein and its lipidic fraction. Furthermore, the treatment of the cells with sublethal concentrations of HNE resulted in up-regulation of AKR1C15, which was partially abrogated by the ROS inhibitors. Collectively, these data indicate an anti-atherogenic function of AKR1C15 through the protection of endothelial cells from damage elicited by toxic lipids such as HNE.     

Oxidized low-density lipoprotein and 15-deoxy-delta 12,14-PGJ2 increase mitochondrial complex I activity in endothelial cells

Oxidized lipids are capable of initiating diverse cellular responses through both receptor-mediated mechanisms and direct posttranslational modification of proteins. Typically, exposure of cells to low concentrations of oxidized lipids induces cytoprotective pathways, whereas high concentrations result in apoptosis. Interestingly, mitochondria can contribute to processes that result in either cytoprotection or cell death. The role of antioxidant defenses such as glutathione in adaptation to stress has been established, but the potential interaction with mitochondrial function is unknown and is examined in this article. Human umbilical vein endothelial cells (HUVEC) were exposed to oxidized LDL (oxLDL) or the electrophilic cyclopentenone 15-deoxy-Delta 12,14-PGJ2 (15d-PGJ2). We demonstrate that complex I activity, but not citrate synthase or cytochrome-c oxidase, is significantly induced by oxLDL and 15d-PGJ2. The mechanism is not clear at present but is independent of the induction of GSH, peroxisome proliferator-activated receptor (PPAR)-gamma, and PPAR-alpha. This response is dependent on the induction of oxidative stress in the cells because it can be prevented by nitric oxide, probucol, and the SOD mimetic manganese(III) tetrakis(4-benzoic acid) porphyrin chloride. This increased complex I activity appears to contribute to protection against apoptosis induced by 4-hydroxynonenal.     

Protective effects of 15-deoxy-Delta12,14-prostaglandin J2 against glutamate-induced cell death in primary cortical neuron cultures: induction of adaptive response and enhancement of cell tolerance primarily through up-regulation of cellular glutathione

There is increasing evidence to suggest that reactive oxygen species, including a variety of lipid oxidation products and other physiologically existing oxidative stimuli, can induce an adaptive response and enhance cell tolerance. In the present study, by using cultured cortical neurons, we investigated the effect of electrophilic lipids, such as 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and 4-hydroxy-2-nonenal (4-HNE) against the cell death induced by H(2)O(2) and glutamate. Pre-treatment with both 15d-PGJ(2) and 4-HNE at sublethal concentrations resulted in a significant protective effect against oxidative stress, and 15d-PGJ(2), in particular, exhibited a complete protective effect against glutamate-induced neuronal cell death. Pre-treatment with 15d-PGJ(2) increased the intracellular glutathione (GSH) as well as the gene expression of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis. 15d-PGJ(2) protected cells from glutamate-induced GSH depletion, while the inhibition of cellular GSH synthesis by buthionine sulfoximine abolished the adaptive response induced by 15d-PGJ(2). These findings indicate that at low levels, 15d-PGJ(2) acts as a potent survival mediator against glutamate-induced insults via the induction of an adaptive response primarily through the up-regulation of the intracellular GSH synthesis.     

The glutathionylation of p65 modulates NF-κB activity in 15-deoxy-Δ¹²,¹⁴-prostaglandin J₂-treated endothelial cells

Protein glutathionylation is a posttranslational modification of cysteine residues with glutathione in response to mild oxidative stress. Because 15-deoxy-Δ12,14-prostaglandin J(2) (15d-PGJ(2)) is an electrophilic prostaglandin that can increase glutathione (GSH) levels and augment reactive oxygen species (ROS) production, we hypothesized that it induces NF-κB-p65 glutathionylation and would exert anti-inflammatory effects. Herein, we show that 15d-PGJ(2) suppresses the expression of ICAM-1 and NF-κB-p65 nuclear translocation. 15d-PGJ(2) upregulates the Nrf2-related glutathione synthase gene and thereby increases the GSH levels. Consistent with this, Nrf2 siRNA molecules abolish the inhibition of p65 nuclear translocation in 15d-PGJ(2)-induced endothelial cells (ECs). ECs treated with GSSG show increased thiol modifications of p65 and also a block in TNFα-induced p65 nuclear translocation and ICAM-1 expression, but not in IκBα degradation. However, the overexpression of glutaredoxin 1 was found to be accompanied by a modest increase in NF-κB activity. Furthermore, we found that multiple cysteine residues in p65 are responsible for glutathionylation. 15d-PGJ(2) was observed to induce p65 glutathionylation and is suppressed by a GSH synthesis inhibitor, buthionine sulfoximine, by catalase, and by Nrf2 siRNA molecules. Our results thus indicate that the GSH/ROS-dependent glutathionylation of p65 is likely to be responsible for 15d-PGJ(2)-mediated NF-κB inactivation and for the enhanced inhibitory effects of 15d-PGJ(2) on TNFα-treated ECs.     

Addition of electrophilic lipids to actin alters filament structure

Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and PGA(1) in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA(1) and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ(2) or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ(2) at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles.     

Induction of endothelial cell apoptosis by lipid hydroperoxide-derived bifunctional electrophiles

Endothelial dysfunction is considered to be the earliest event in atherogenesis. Oxidative stress, inflammation, and apoptosis play critical roles in its progression and onset. Lipid peroxidation, which occurs during oxidative stress, results in the formation of lipid hydroperoxide-derived bifunctional electrophiles such as 4-hydroxy-2(E)-nonenal that induce apoptosis. In this study, recently identified lipid hydroperoxide-derived bifunctional electrophiles 4-oxo-2(E)-nonenal (ONE; 5-30 microm) and 4,5-epoxy-2(E)-decenal (EDE; 10-20 microM) were shown to cause a dose- and time-dependent apoptosis in EA.hy 926 endothelial cells. This was manifest by morphological changes, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. Bifunctional electrophiles caused cytochrome c release from mitochondria into the cytosol, implicating a mitochondrial pathway of apoptosis in the endothelial cells. The novel carboxylate-containing lipid hydroperoxide-derived bifunctional electrophile 9,12-dioxo-10(E)-dodecenoic acid was inactive because it could not translocate across the plasma membrane. However, its less polar methyl ester derivative (2-10 microM) was the most potent inducer of apoptosis of any bifunctional electrophile that has been tested. An acute decrease in intracellular glutathione (GSH) preceded the onset of apoptosis in bifunctional electrophile-treated cells. The ability of ONE and EDE to deplete GSH was directly correlated with their predicted reactivity toward nucleophilic amino acids. Liquid chromatography/mass spectrometry methodology was developed in order to examine the intracellular and extracellular concentrations of bifunctional electrophile-derived GSH adducts. Relative intracellular/extracellular ratios of the GSH adducts were identical with the rank order of potency for inducing caspase 3 activation. This suggests that there may be a role for the bifunctional electrophile-derived GSH adducts in the apoptotic response. N-Acetylcysteine rescued bifunctional electrophile-treated cells from apoptosis, whereas the GSH biosynthesis inhibitor d,l-buthionine-(R,S)-sulfoximine sensitized the cells to apoptosis. These data suggest that lipid hydroperoxide-derived bifunctional electrophiles may play an important role in cardiovascular pathology through their ability to induce endothelial cell apoptosis.     

Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-Δ-prostaglandin J2

Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 μM concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexes is evident when Drp1 is incubated with a non-cleavable GTP analog, GTPγS or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration.     

4-Hydroxynonenal and PPARgamma ligands affect proliferation, differentiation, and apoptosis in colon cancer cells

PPARgamma ligands inhibit growth and induce apoptosis of various cancer cells. 4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation and induces differentiation or apoptosis in neoplastic cells. The aim of this work was to investigate the effects of PPARgamma ligands (rosiglitazone and 15-deoxy-prostaglandin J2 (15d-PGJ2)) and HNE, alone or in association, on proliferation, apoptosis, differentiation, and growth-related and apoptosis-related gene expression in colon cancer cells (CaCo-2 cells). PPARgamma ligands inhibited cell proliferation (IC50 was 37.47+/-6.6 microM, for 15d-PGJ2, and 170.34+/-20 microM for rosiglitazone). HNE (1 microM) inhibited cell growth by 70%. Apoptosis was induced by 15d-PGJ2 and HNE and, to a minor extent, rosiglitazone. Differentiation was induced by rosiglitazone and by 15d-PGJ2, but not by HNE. PPARgamma ligands inhibited c-myc expression. HNE induced a transitory increase in c-myc expression and a subsequent down-regulation. HNE induced p21 expression, whereas PPARgamma ligands did not. Expression of the bax gene was increased by HNE and 15d-PGJ2, but not by rosiglitazone. No synergism or antagonism was found between HNE and PPARgamma ligands. Both apoptosis and differentiation induction may be responsible for the inhibition of proliferation by PPARgamma ligands; apoptosis and c-myc and p21 expression seem to be involved in the inhibition of proliferation by HNE.     

15-Deoxy-Delta(12,14)-prostaglandin J(2) suppresses IL-6-induced STAT3 phosphorylation via electrophilic reactivity in endothelial cells

In this study, the effects of 15d-PGJ(2) were investigated in IL-6-activated endothelial cells (ECs). 15d-PGJ(2) was found to abrogate phosphorylation on tyr705 of STAT3 in IL-6-treated ECs, in a dose- and time-dependent manner, but did not inhibit serine phosphorylation of STAT3 and the upperstream JAK2 phosphorylation. Other PPAR activators, such as WY1643 or ciglitazone, had no effect upon IL-6-induced STAT3 phosphorylation. Additionally, neither orthovanadate nor l-NAME treatment reverses the inhibition of STAT3 phosphorylation by 15d-PGJ(2). Otherwise, the effect of 15d-PGJ(2) requires the alpha,beta-unsaturated carbonyl group in the cyclopentane ring. A 15d-PGJ(2) analog, 9,10-Dihydro-15d-PGJ(2), which lack alpha,beta-unsaturated carbonyl group showed no increase in ROS production and no effect in inhibition of IL-6-induced STAT3 phosphorylation. The electrophilic compound, acrolein, mimics the inhibition effect of 15d-PGJ(2). Among the antioxidants, only NAC and glutathione reversed the effects of 15d-PGJ(2). NAC, glutathione and DTT all reversed the inhibition of STAT3 phosphorylation when preincubated with 15d-PGJ(2). The inhibition of ICAM-1 gene expression by 15d-PGJ(2) was abrogated by NAC and glutathione in IL-6-treated ECs. Taken together, these results suggest that 15d-PGJ(2) inhibits IL-6-stimulated phosphorylation on tyr705 of STAT3 dependent on its own electrophilic reactivity in ECs.     

Oxidative modifications of cardiac mitochondria and inhibition of cytochrome c oxidase activity by 4-hydroxynonenal

4-hydroxynonenal (HNE) is a highly toxic product of lipid peroxidation (LPO). Its role in the inhibition of cytochrome c oxidase activity and oxidative modifications of mitochondrial lipids and proteins were investigated. The exposure of mitochondria isolated from rat heart to HNE resulted in a time- and concentration-dependent inhibition of cytochrome c oxidase activity with an IC50 value of 8.3 +/- 1.0 microM. Immunoprecipitation-Western blot analysis showed the formation of HNE adducts with cytochrome c oxidase subunit I. The loss of cytochrome c oxidase activity was also accompanied by reduced thiol group content and increased HNE-lysine fluorescence. Furthermore, there was a marked increase in conjugated diene formation indicating LPO induction by HNE. Fluorescence measurements revealed the formation of bityrosines and increased surface hydrophobicity of HNE-treated mitochondrial membranes. Superoxide dismutase + catalase and the HO* radical scavenger mannitol partially prevented inhibition of cytochrome c oxidase activity and formation of bityrosines. These findings suggest that HNE induces formation of reactive oxygen species and its damaging effect on mitochondria involves both formation of HNE-protein adducts and oxidation of membrane lipids and proteins by free radicals.     

Induction of apoptosis by 15d-PGJ2 via ROS formation: an alternative pathway without PPARγ activation in non-small cell lung carcinoma A549 cells

Cyclopentenone prostaglandin 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), which is generated from the dehydration of PGD(2), is a natural ligand of peroxisome proliferator-activated receptor gamma (PPARγ) and a potential apoptotic mediator. The synthetic PPARγ ligands, troglitazone and ciglitazone, inhibit tumor progression in many cells by PPARγ activation, but the mechanism of 15d-PGJ(2) is still unclear. In this study, GW9662, an antagonist of PPARγ, and quercetin, a natural antioxidant, were used to study the apoptotic mechanism of 15d-PGJ(2) in A549 cells. Results showed that 15d-PGJ(2) induced apoptosis, which was associated with the production of reactive oxygen species (ROS) and the decrease of GSH levels. Furthermore, quercetin reduced the activity of caspases in 15d-PGJ(2)-induced apoptotic processes. These results suggest that 15d-PGJ(2) induces apoptosis in A549 cells mainly through the formation of ROS; it does not depend on PPARγ activation. Moreover, these findings support the use of quercetin and PPARγ agonists in non-small cell lung carcinoma.     

Electrophilic Aldehydes Generated by Sperm Metabolism Activate Mitochondrial Reactive Oxygen Species Generation and Apoptosis by Targeting Succinate Dehydrogenase

Oxidative stress is a major cause of defective sperm function in cases of male infertility. Such stress is known to be associated with high levels of superoxide production by the sperm mitochondria; however, the causes of this aberrant activity are unknown. Here we show that electrophilic aldehydes such as 4-hydroxynonenal (4HNE) and acrolein, generated as a result of lipid peroxidation, target the mitochondria of human spermatozoa and stimulate mitochondrial superoxide generation in a dose- and time-dependent manner. The activation of mitochondrial electron leakage by 4HNE is shown to involve the disruption of succinate dehydrogenase activity and subsequent activation of an intrinsic apoptotic cascade beginning with a loss of mitochondrial membrane potential and terminating in oxidative DNA adduct formation, DNA strand breakage, and cell death. A tight correlation between spontaneous mitochondrial superoxide generation and 4HNE content (R² = 0.89) in untreated populations of human spermatozoa emphasized the pathophysiological significance of these findings. The latter also provide a biochemical explanation for the self-perpetuating nature of oxidative stress in the male germ line, with the products of lipid peroxidation stimulating free radical generation by the sperm mitochondria in a positive feedback loop.     

Opposite effects of prostaglandin-J2 on VEGF in normoxia and hypoxia: role of HIF-1

The vascular endothelial growth factor (VEGF) is produced in response to hypoxia or inflammatory cytokines. In normoxia VEGF synthesis is upregulated by 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) via induction of heme oxygenase-1 (HO-1). Here we compared the influence of 15d-PGJ(2) on VEGF expression in human microvascular endothelial cells in normoxia (approximately 20% O(2)) and hypoxia ( approximately 2% O(2)). Regardless of the oxygen concentration, 15d-PGJ(2) inhibited activity of hypoxia inducible factor-1 (HIF-1), the major hypoxic regulator of VEGF. However, in normoxic conditions 15d-PGJ(2) (1-10microM) activated the VEGF promoter and increased synthesis of the VEGF protein. Concomitantly, it strongly induced expression of HO-1. In contrast, in hypoxia, 15d-PGJ(2) decreased VEGF promoter activity and reduced VEGF release by 50%. Inhibition of HO-1 activity additionally attenuated VEGF synthesis in hypoxia. We conclude that induction of HO-1 by 15d-PGJ(2) results in augmentation of VEGF synthesis in normoxia. In hypoxia, however, the stimulatory effect of HO-1 is outweighed by 15d-PGJ(2)-mediated inhibition of the HIF-1 pathway.     

Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1

We showed earlier that 15 deoxy Δ^12,14 prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion [1]. However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria by 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity.     

Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential

Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of ROS (reactive oxygen species). This proton leak can be induced by the lipid peroxidation products of ROS, such as HNE (4-hydroxynonenal). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and ANT (adenine nucleotide translocase), thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We have found that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent on incubation with respiratory substrate. In the presence of HNE, mitochondria energized with succinate became progressively more leaky to protons over time compared with mitochondria in the absence of either HNE or succinate. Energized mitochondria must attain a high membrane potential to allow HNE to activate uncoupling: a drop of 10-20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), ANT (64%) and other pathways (25%). Our findings have shown that exogenous HNE only activates uncoupling at high membrane potential. These results suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production.     

Oxidative modifications of cardiac mitochondria and inhibition of cytochrome c oxidase activity by 4-hydroxynonenal

Abstract

4-Hydroxynonenal (HNE) is a highly toxic product of lipid peroxidation (LPO). Its role in the inhibition of cytochrome c oxidase activity and oxidative modifications of mitochondrial lipids and proteins were investigated. The exposure of mitochondria isolated from rat heart to HNE resulted in a time- and concentration-dependent inhibition of cytochrome c oxidase activity with an IC₅₀ value of 8.3 ± 1.0 μM. Immunoprecipitation-Western blot analysis showed the formation of HNE adducts with cytochrome c oxidase subunit I. The loss of cytochrome c oxidase activity was also accompanied by reduced thiol group content and increased HNE-lysine fluorescence. Furthermore, there was a marked increase in conjugated diene formation indicating LPO induction by HNE. Fluorescence measurements revealed the formation of bityrosines and increased surface hydrophobicity of HNE-treated mitochondrial membranes. Superoxide dismutase + catalase and the HO^? radical scavenger mannitol partially prevented inhibition of cytochrome c oxidase activity and formation of bityrosines. These findings suggest that HNE induces formation of reactive oxygen species and its damaging effect on mitochondria involves both formation of HNE–protein adducts and oxidation of membrane lipids and proteins by free radicals.     

Protective role of glutathione and glutathione transferases in mutagenesis and carcinogenesis

Glutathione (GSH) alone detoxifies electrophiles with an effectiveness which depends on the rate of the reaction and the concentration of GSH. If electrophiles are substrates for GSH transferase isoenzymes, the effectiveness of detoxication is much enhanced due to the increased rate of reaction and it is also independent of GSH concentration to low levels of GSH depletion, since the Km for GSH is approximately 0.1 mM. In this paper detoxication of electrophilic metabolites of the hepatocarcinogen N-methyl-4-aminoazobenzene which are not substrates for GSH transferases and the carcinogenic electrophile derived from the hepatocarcinogen aflatoxin B1 which is a poor substrate is compared with detoxication of electrophiles which are good substrates and which although bacterial mutagens are not carcinogenic in organs containing the appropriate GSH transferases. GSH transferases detoxify not only electrophiles derived from xenobiotics, but also endogenous electrophiles which are usually the consequence of free radical damage in the presence of oxygen to lipids and DNA and include lipid and DNA hydroperoxides and alkenals arising from the decomposition of lipid hydroperoxides. Studies in the rat and other mammals show the GSH transferases to be dimers in which the subunits are members of a gene super-family. There are three, perhaps four multigene families namely, alpha containing subunits 1, 2, 8 and 10; mu containing subunits 3, 4, 6 and 9; pi containing subunit 7 and subunits 5 and 5* which are so far unassigned. Subunit 5* is apparently restricted to the nucleus and is noteworthy for its activity towards DNA hydroperoxides. Studies in the human are not as advanced as in the rat but so far reveal close similarities. The ability of GSH transferases to detoxify electrophiles is important in carcinogenesis at a number of points. They may inhibit initiation and tumour proportion, but they may be advantageous to the developing tumour cell, and may be acquired in increased amounts during malignant progression. In many tumour cells the development of lines resistant to anticancer drugs is associated with an increased expression of GSH transferases, particularly GSH transferase pi in human cells.     

Thioredoxin as a molecular target of cyclopentenone prostaglandins

Prostaglandin (PG) D2, a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield the bioactive cyclopentenone-type PGs of the J2 series, such as 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2). We have shown previously that 15d-PGJ2 is a potent electrophile that causes intracellular oxidative stress and redox alteration in human neuroblastoma SH-SY5Y cells. In the present study, based on the observation that the electrophilic center of 15d-PGJ2 was involved in the pro-oxidant effect, we investigated the role of thioredoxin 1 (Trx), an endogenous redox regulator, against 15d-PGJ2-induced oxidative cell injury. It was observed that the 15d-PGJ2-induced oxidative stress was significantly suppressed by the Trx overexpression. In addition, the treatment of SH-SY5Y cells with biotinylated 15d-PGJ2 resulted in the formation of a 15d-PGJ2-Trx adduct, indicating that 15d-PGJ2 directly modified the endogenous Trx in the cells. To further examine the mechanism of the 15d-PGJ2 modification of Trx, human recombinant Trx treated with 15d-PGJ2 was analyzed by mass spectrometry. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the 15d-PGJ2-treated human recombinant Trx demonstrated the addition of one molecule of 15d-PGJ2 per protein molecule. Moreover, the electrospray ionization-liquid chromatography/mass spectrometry/mass spectrometry analysis identified two cysteine residues, Cys-35 and Cys-69, as the targets of 15d-PGJ2. These residues may represent the direct sensors of the electrophilic PGs that induce the intracellular redox alteration and neuronal cell death.