Scavengers of reactive γ-ketoaldehydes extend Caenorhabditis elegans lifespan and healthspan through protein-level interactions with SIR-2.1 and ETS-7.

Isoketals (IsoKs) are highly reactive γ-ketoaldehyde products of lipid peroxidation that covalently adduct lysine side chains in proteins, impairing their function. Using C. elegans as a model organism, we sought to test the hypothesis that IsoKs contribute to molecular aging through adduction and inactivation of specific protein targets, and that this process can be abrogated using salicylamine (SA), a selective IsoK scavenger. Treatment with SA extends adult nematode longevity by nearly 56% and prevents multiple deleterious age-related biochemical and functional changes. Testing of a variety of molecular targets for SA's action revealed the sirtuin SIR-2.1 as the leading candidate. When SA was administered to a SIR-2.1 knockout strain, the effects on lifespan and healthspan extension were abolished. The SIR-2.1-dependent effects of SA were not mediated by large changes in gene expression programs or by significant changes in mitochondrial function. However, expression array analysis did show SA-dependent regulation of the transcription factor ets-7 and associated genes. In ets-7 knockout worms, SA's longevity effects were abolished, similar to sir-2.1 knockouts. However, SA dose-dependently increases ets-7 mRNA levels in non-functional SIR-2.1 mutant, suggesting that both are necessary for SA's complete lifespan and healthspan extension.

Cardiolipin fatty acid remodeling regulates mitochondrial function by modifying the electron entry point in the respiratory chain.

Modifications of cardiolipin (CL) levels or compositions are associated with changes in mitochondrial function in a wide range of pathologies. We have made the discovery that acetaminophen remodels CL fatty acids composition from tetralinoleoyl to linoleoyltrioleoyl-CL, a remodeling that is associated with decreased mitochondrial respiration. Our data show that CL remodeling causes a shift in electron entry from complex II to the ß-oxidation electron transfer flavoprotein quinone oxidoreductase (ETF/QOR) pathway. These data demonstrate that electron entry in the respiratory chain is regulated by CL fatty acid composition and provide proof-of-concept that pharmacological intervention can be used to modify CL composition.

Measurement of F2- isoprostanes and isofurans using gas chromatography-mass spectrometry.

F2-Isoprostanes (IsoPs) are isomers of prostaglandin F2α formed from the nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. Since discovery of these molecules by Morrow and Roberts in 1990, F2-IsoPs have been shown to be excellent biomarkers as well as potent mediators of oxidative stress in vivo in humans. Isofurans (IsoFs) are also oxidation products generated from the nonenzymatic oxidation of arachidonic acid. IsoFs are preferentially formed instead of F2-IsoPs in settings of increased oxygen tension. The protocol presented herein is the current methodology that our laboratory uses to quantify F2-IsoPs and IsoFs in biological tissues and fluids using gas chromatography/mass spectrometry (GC/MS). A variety of analytical procedures to measure F2-IsoPs, including other GC/MS methods and liquid chromatography/MS and immunological approaches, are reported in the literature. This method provides a very low limit of quantitation and is suitable for analysis of both F2-IsoPs and IsoFs from a variety of biological sources including urine, plasma, tissues, cerebral spinal fluid, exhaled breath condensate, and amniotic fluid, among others.

Acetaminophen inhibits cytochrome c redox cycling induced lipid peroxidation.

Cytochrome (cyt) c can uncouple from the respiratory chain following mitochondrial stress and catalyze lipid peroxidation. Accumulating evidence shows that this phenomenon impairs mitochondrial respiratory function and also initiates the apoptotic cascade. Therefore, under certain conditions a pharmacological approach that can inhibit cyt c catalyzed lipid peroxidation may be beneficial. We recently showed that acetaminophen (ApAP) at normal pharmacologic concentrations can prevent hemoprotein-catalyzed lipid peroxidation in vitro and in vivo by reducing ferryl heme to its ferric state. We report here, for the first time, that ApAP inhibits cytochrome c-catalyzed oxidation of unsaturated free fatty acids and also the mitochondrial phospholipid, cardiolipin. Using isolated mitochondria, we also showed that ApAP inhibits cardiolipin oxidation induced by the pro-apoptotic protein, tBid. We found that the IC(50) of the inhibition of cardiolipin oxidation by ApAP is similar in both intact isolated mitochondria and cardiolipin liposomes, suggesting that ApAP penetrates well into the mitochondria. Together with our previous results, the findings presented herein suggest that ApAP is a pleiotropic inhibitor of peroxidase catalyzed lipid peroxidation. Our study also provides a potentially novel pharmacological approach for inhibiting the cascade of events that can result from redox cycling of cyt c.

Effects of age and caloric restriction on lipid peroxidation: measurement of oxidative stress by F2-isoprostane levels.

The free radical theory of aging proposes that the accumulation of oxidative damage is a key component of the aging process. The discovery of F2-isoprostanes (F2-isoPs) and their establishment as a sensitive and accurate biomarker of lipid peroxidation represents a major advance for measuring the oxidative stress status of an organism. We have shown that plasma free and total (free plus esterified) F2-isoPs increase with age (185% and 66%, respectively), and that these increases are reduced by life-extending caloric restriction (50% and 23%, respectively). In addition, we found that levels of esterified F2-isoPs increase 68% with age in liver, and 76% with age in kidney. Caloric restriction modulated the age-related increase, reducing the esterified F2-isoPs levels 27% in liver and 35% in kidney. These age-related increases in esterified F2-isoPs levels correlate well with DNA oxidation, as measured by 8-oxodeoxyguanosine production demonstrating that F2-isoPs are an excellent biomarker for age-related changes in oxidative damage to membranes.

Quantification of F-ring isoprostane-like compounds (F4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a stable isotope dilution mass spectrometric assay.

Lipid peroxidation has been implicated in the pathophysiological sequelae of human neurodegenerative disorders. It is recognized that quantification of lipid peroxidation is best assessed in vivo by measuring a series of prostaglandin (PG) F2-like compounds termed F2-isoprostanes (IsoPs) in tissues in which arachidonic acid is abundant. Unlike other organs, the major polyunsaturated fatty acid (PUFA) in the brain is docosahexaenoic acid (DHA, C22:6 omega-6), and this fatty acid is particularly enriched in neurons. We have previously reported that DHA undergoes oxidation in vitro and in vivo resulting in the formation of a series of F2-IsoP-like compounds termed F4-neuroprostanes (F4-NPs). We recently chemically synthesized one F4-NP, 17-F4c-NP, converted it to an 18O-labeled derivative, and utilized it as an internal standard to develop an assay to quantify endogenous production of F4-NPs by gas chromatography (GC)/negative ion chemical ionization (NICI) mass spectrometry (MS). The assay is highly precise and accurate. The lower limit of sensitivity is approximately 10 pg. Levels of F4-NPs in brain tissue from rodents were 8.7 +/- 2.0 ng/g wet weight (mean +/- S.D.). Levels of the F4-NPs in brains from normal humans were found to be 4.9 +/- 0.6 ng/g (mean +/- S.D.) and were 2.1-fold higher in affected regions of brains from humans with Alzheimer's disease (P = 0.02). Thus, this assay provides a sensitive and accurate method to assess oxidation of DHA in animal and human tissues and will allow for the further elucidation of the role of oxidative injury to the central nervous system in association with human neurodegenerative disorders.

Formation of prostaglandins E2 and D2 via the isoprostane pathway: a mechanism for the generation of bioactive prostaglandins independent of cyclooxygenase.

It has heretofore been assumed that the cyclooxygenases (COXs) are solely responsible for peostaglandin (PG) synthesis in vivo. An important structural feature of PGH2 formed by COX is the trans-configuration of side chains relative to the prostane ring. Previously, we reported that a series of PG-like compounds termed isoprostanes (IsoPs) are formed in vivo in humans from the free radical-catalyzed peroxidation of arachidonate independent of COX. A major difference between these compounds and PGs is that IsoPs are formed from endoperoxide intermediates, the vast majority of which contain side chains that are cis relative to the prostane ring. In addition, unlike the formation of eicosanoids from COX, IsoPs are formed as racemic mixtures because they are generated nonenzymatically. IsoPs containing E- and D-type prostane rings (E2/D2-IsoPs) are one class of IsoPs formed, and we have reported previously that one of the major IsoPs generated is 15-E2t-IsoP (8-iso-PGE2). Unlike PGE2, 15-E2t-IsoP is significantly more unstable in buffered solutions in vitro and undergoes epimerization to PGE2. Analogously, the D-ring IsoP (15-D2c-IsoP) would be predicted to rearrange to PGD2. We now report that compounds identical in all respects to PGE2 and PGD2 and their respective enantiomers are generated in vivo via the IsoP pathway, presumably by epimerization of racemic 15-E2t-IsoP and 15-D2c-IsoP, respectively. Racemic PGE2 and PGD2 were present esterified in phospholipids derived from liver tissue from rats exposed to oxidant stress at levels of 24 +/- 16 and 37 +/- 12 ng/g of tissue, respectively. In addition, racemic PGs, particularly PGD2, were present unesterified in urine from normal animals and humans and represented up to 10% of the total PG detected. Levels of racemic PGD2 increased 35-fold after treatment of rats with carbon tetrachloride to induce oxidant stress. In this setting, PGD2 and its enantiomer generated by the IsoP pathway represented approximately 30% of the total PGD2 present in urine. These findings strongly support the contention that a second pathway exists for the formation of bioactive PGs in vivo that is independent of COX.

Human colorectal cancer cells efficiently conjugate the cyclopentenone prostaglandin, prostaglandin J(2), to glutathione.

Cyclopentenone prostaglandins (PGs), particularly those of the J-series, affect proliferation and differentiation in a number of cell lines. J-ring PGs have been shown to be ligands for the peroxisome proliferator-activated receptor (PPAR)-gamma and to modulate NF-kappaB-mediated gene transcription. We have previously reported that large quantities of eicosanoids, including PGJ(2), are produced by the human colorectal cancer cell line HCA-7 while lesser amounts of Delta(12)-PGJ(2) and 15-deoxy-Delta(12,14)-PGJ(2) are formed. In this and other cell lines, cyclopentenone PGs have been shown to increase cell proliferation, but factors that influence their formation and metabolism are poorly understood. Unlike other PGs, cyclopentenone PGs contain alpha,beta-unsaturated carbonyl groups that readily adduct various biomolecules such as glutathione (GSH) in vitro. We now report that in HCA-7 cells, PGJ(2) is largely metabolized by conjugation to GSH. Characterization of the adducts by liquid chromatography (LC)-mass spectrometry (MS) revealed two major metabolites consisting of (1) a novel GSH conjugate in which the carbonyl at C-11 of PGJ(2) is reduced and (2) intact PGJ(2) conjugated to GSH. Approximately 70% of the PGJ(2) added to HCA-7 cells was esterifed to GSH after 2 h of incubation, suggesting this pathway represents the major route of metabolic disposition of PGJ(2) in HCA-7 cells.

Formation of highly reactive A-ring and J-ring isoprostane-like compounds (A4/J4-neuroprostanes) in vivo from docosahexaenoic acid.

Free radical-initiated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. Docosahexaenoic acid is the most abundant unsaturated fatty acid in the central nervous system. We have shown previously that this 22-carbon fatty acid can yield, upon oxidation, isoprostane-like compounds termed neuroprostanes, with E/D-type prostane rings (E(4)/D(4)-neuroprostanes). Eicosanoids with E/D-type prostane rings are unstable and dehydrate to cyclopentenone-containing compounds possessing A-type and J-type prostane rings, respectively. We thus explored whether cyclopentenone neuroprostanes (A(4)/J(4)-neuroprostanes) are formed from the dehydration of E(4)/D(4)-neuroprostanes. Indeed, oxidation of docosahexaenoic acid in vitro increased levels of putative A(4)/J(4)-neuroprostanes 64-fold from 88 +/- 43 to 5463 +/- 2579 ng/mg docosahexaenoic acid. Chemical approaches and liquid chromatography/electrospray ionization tandem mass spectrometry definitively identified them as A(4)/J(4)-neuroprostanes. We subsequently showed these compounds are formed in significant amounts from a biological source, rat brain synaptosomes. A(4)/J(4)-neuroprostanes increased 13-fold, from a basal level of 89 +/- 72 ng/mg protein to 1187 +/- 217 ng/mg (n = 4), upon oxidation. We also detected these compounds in very large amounts in fresh brain tissue from rats at levels of 97 +/- 25 ng/g brain tissue (n = 3) and from humans at levels of 98 +/- 26 ng/g brain tissue (n = 5), quantities that are nearly an order of magnitude higher than other classes of neuroprostanes. Because of the fact that A(4)/J(4)-neuroprostanes contain highly reactive cyclopentenone ring structures, it would be predicted that they readily undergo Michael addition with glutathione and adduct covalently to proteins. Indeed, incubation of A(4)/J(4)-neuroprostanes in vitro with excess glutathione resulted in the formation of large amounts of adducts. Thus, these studies have identified novel, highly reactive A/J-ring isoprostane-like compounds that are derived from docosahexaenoic acid in vivo.