Triphenylphosphonium (TPP)-Based Antioxidants: A New Perspective on Antioxidant Design

Mitochondrial oxidative damage and dysfunction contribute to a wide range of human diseases. Considering the limitation of conventional antioxidants and that mitochondria are the main source of reactive oxygen species (ROS) which induce oxidative damage, mitochondria-targeted antioxidants which can selectively block mitochondrial oxidative damage and prevent various types of cell death have been widely developed. As a lipophilic cation, triphenylphosphonium (TPP) has been commonly used in designing mitochondria-targeted antioxidants. Conjugated with the TPP moiety, antioxidants can achieve more than 1000-fold higher mitochondrial concentration depending on cell membrane potentials and mitochondrial membrane potentials. Herein we discuss the deficiencies of conventional antioxidants and the advantages of mitochondrial targeting, and review various types of TPP-based mitochondria-targeted antioxidants. These provide theoretical and background support for the design of new anti-oxidant.     

A Prototypical Small‐Molecule Modulator Uncouples Mitochondria in Response to Endogenous Hydrogen Peroxide Production

A high membrane potential across the mitochondrial inner membrane leads to the production of the reactive oxygen species (ROS) implicated in aging and age‐related diseases. A prototypical drug for the correction of this type of mitochondrial dysfunction is presented. MitoDNP‐SUM accumulates in mitochondria in response to the membrane potential due to its mitochondria‐targeting alkyltriphenylphosphonium (TPP) cation and is uncaged by endogenous hydrogen peroxide to release the mitochondrial uncoupler, 2,4‐dinitrophenol (DNP). DNP is known to reduce the high membrane potential responsible for the production of ROS. The approach potentially represents a general method for the delivery of drugs to the mitochondrial matrix through mitochondria targeting and H₂O₂‐induced uncaging.     

Modulation of Cellular Fate of Vinyl Triarylamines through Structural Fine Tuning: To Stay or Not To Stay in the Mitochondria?

Mitochondria are involved in many cellular pathways and dysfunctional mitochondria are linked to various diseases. Hence efforts have been made to design mitochondria-targeted fluorophores for monitoring the mitochondrial status. However, the factors that govern the mitochondria-targeted potential of dyes are not well-understood. In this context, we synthesized analogues of the TP-2Bzim probe belonging to the vinyltriphenylamine (TPA) class and already described for its capacity to bind nuclear DNA in fixed cells and mitochondria in live cells. These analogues ( TP-1Bzim, TPⁿ-2Bzim, TP¹⁺-2Bzim, TN-2Bzim ) differ in the cationic charge, the number of vinylbenzimidazolium branches and the nature of the triaryl core. Using microscopy, we demonstrated that the cationic derivatives accumulate in mitochondria but do not reach mtDNA. Under depolarisation of the mitochondrial membrane, TP-2Bzim and TP¹⁺-2Bzim translocate to the nucleus in direct correlation with their strong DNA affinity. This reversible phenomenon emphasizes that these probes can be used to monitor ΔΨ_m variations.     

Mitochondrial Membrane Disrupting Molecules for Selective Killing of Senescent Cells

Cellular senescence, a stable form of cell cycle arrest, facilitates protection from tumorigenesis and aids in tissue repair as they accumulate in the body at an early age. However, long-term retention of senescent cells causes inflammation, aging of the tissue, and progression of deadly diseases such as obesity, diabetes, and atherosclerosis. Various attempts have been made to achieve selective elimination of senescent cells from the body, yet little has been explored in designing the mitochondria-targeted senolytic agent. Many characteristics of senescence are associated with mitochondria. Here we have designed a library of alkyl-monoquaternary ammonium-triphenyl phosphine (TPP) and alkyl-diquaternary ammonium-TPP of varying alkyl chain lengths, which target the mitochondria; we also studied their senolytic properties. It was observed that the alkyl-diquaternary ammonium-TPP with the longest chain length induced apoptosis in senescent cells selectively via an increase of reactive oxygen species (ROS) and mitochondrial membrane disruption. This study demonstrates that mitochondria could be a potential target for designing new small molecules as senolytic agents for the treatment of a variety of dysfunctions associated with pathological aging.     

Treatment Strategies that Enhance the Efficacy and Selectivity of Mitochondria-Targeted Anticancer Agents

Nearly a century has passed since Otto Warburg first observed high rates of aerobic glycolysis in a variety of tumor cell types and suggested that this phenomenon might be due to an impaired mitochondrial respiratory capacity in these cells. Subsequently, much has been written about the role of mitochondria in the initiation and/or progression of various forms of cancer, and the possibility of exploiting differences in mitochondrial structure and function between normal and malignant cells as targets for cancer chemotherapy. A number of mitochondria-targeted compounds have shown efficacy in selective cancer cell killing in pre-clinical and early clinical testing, including those that induce mitochondria permeability transition and apoptosis, metabolic inhibitors, and ROS regulators. To date, however, none has exhibited the standards for high selectivity and efficacy and low toxicity necessary to progress beyond phase III clinical trials and be used as a viable, single modality treatment option for human cancers. This review explores alternative treatment strategies that have been shown to enhance the efficacy and selectivity of mitochondria-targeted anticancer agents in vitro and in vivo, and may yet fulfill the clinical promise of exploiting the mitochondrion as a target for cancer chemotherapy.     

PEG35 as a Preconditioning Agent against Hypoxia/Reoxygenation Injury

Pharmacological conditioning is a protective strategy against ischemia/reperfusion injury, which occurs during liver resection and transplantation. Polyethylene glycols have shown multiple benefits in cell and organ preservation, including antioxidant capacity, edema prevention and membrane stabilization. Recently, polyethylene glycol 35 kDa (PEG35) preconditioning resulted in decreased hepatic injury and protected the mitochondria in a rat model of cold ischemia. Thus, the study aimed to decipher the mechanisms underlying PEG35 preconditioning-induced protection against ischemia/reperfusion injury. A hypoxia/reoxygenation model using HepG2 cells was established to evaluate the effects of PEG35 preconditioning. Several parameters were assessed, including cell viability, mitochondrial membrane potential, ROS production, ATP levels, protein content and gene expression to investigate autophagy, mitochondrial biogenesis and dynamics. PEG35 preconditioning preserved the mitochondrial function by decreasing the excessive production of ROS and subsequent ATP depletion, as well as by recovering the membrane potential. Furthermore, PEG35 increased levels of autophagy-related proteins and the expression of genes involved in mitochondrial biogenesis and fusion. In conclusion, PEG35 preconditioning effectively ameliorates hepatic hypoxia/reoxygenation injury through the enhancement of autophagy and mitochondrial quality control. Therefore, PEG35 could be useful as a potential pharmacological tool for attenuating hepatic ischemia/reperfusion injury in clinical practice.     

Preparation and characterization of venlafaxine hydrochloride‐loaded chitosan nanoparticles and in vitro release of drug

The venlafaxine hydrochloride (VHL)‐loaded chitosan nanoparticles were prepared by ionic gelation of chitosan (CS) using tripolyphosphate (TPP). The nanoparticles were characterized using FTIR, differential scanning calorimetry, X‐ray diffraction, dynamic light scattering, transmission electron microscopy, and X‐ray photoelectron spectroscopy. The effect of concentration of CS, polyethylene glycol (PEG), VHL and CS/TPP mass ratio on the particle size and zeta potential of nanoparticles was examined. The particle size of CS/TPP nanoparticles and VHL‐loaded CS/TPP nanoparticles was within the range of 200–400 nm with positive surface charge. In the case of VHL‐loaded nanoparticles and PEG‐coated CS/TPP nanoparticles, the particle size increases and surface charge decreases with increasing concentration of VHL and PEG. Both placebo and VHL‐loaded CS/TPP nanoparticles were observed to be spherical in nature. PEG coating on the surface of CS/TPP nanoparticles was confirmed by XPS analysis. Maximum drug entrapment efficiency (70%) was observed at 0.6 mg/mL drug concentration. In vitro drug release study at 37°C ± 0.5°C and pH 7.4 exhibited initial burst release followed by a steady release. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Calcium Ions Regulate K+ Uptake into Brain Mitochondria: The Evidence for a Novel Potassium Channel

The mitochondrial response to changes of cytosolic calcium concentration has a strong impact on neuronal cell metabolism and viability. We observed that Ca²⁺ additions to isolated rat brain mitochondria induced in potassium ion containing media a mitochondrial membrane potential depolarization and an accompanying increase of mitochondrial respiration. These Ca²⁺ effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BK_Ca channel). Furthermore, NS1619 – a BK_Ca channel opener – induced potassium ion–specific effects on brain mitochondria similar to those induced by Ca²⁺. These findings suggest the presence of a calcium-activated, large conductance potassium channel (sensitive to charybdotoxin and NS1619), which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers. The conductance of the reconstituted channel was 265 pS under gradient (50/450 mM KCl) conditions. Its reversal potential was equal to 50 mV, which proved that the examined channel was cation-selective. We also observed immunoreactivity of anti-β₄ subunit (of the BK_Ca channel) antibodies with ~26 kDa proteins of rat brain mitochondria. Immunohistochemical analysis confirmed the predominant occurrence of β₄ subunit in neuronal mitochondria. We hypothesize that the mitochondrial BK_Ca channel represents a calcium sensor, which can contribute to neuronal signal transduction and survival.     

Mitochondria-Targeting Polymer Micelles in Stepwise Response Releasing Gemcitabine and Destroying the Mitochondria and Nucleus for Combined Antitumor Chemotherapy

Mitochondrial DNA and nuclear DNA are essential genetic material which play an important role in maintaining normal metabolism, survival, and proliferation of cells. Constructing a mitochondria-targeting stimuli-responsive nano-drug delivery system releasing chemotherapeutic agents in a stepwise response manner and destroying mitochondrial DNA and nuclear DNA simultaneously is an effective way to improve the anti-tumor effect of chemotherapeutic agents. In this study, a new mitochondria-targeting pH/ROS dual-responsive block copolymer TPP-PEG2k-b-(BS-AA)_n (P1), untargeted pH/ROS dual-responsive copolymer mPEG2k-b-(BS-AA)_n (P2), pH single-responsive copolymer (mPEG2k-b-(AH-AA)_n (P3), ROS single-responsive copolymer mPEG2k-b-(SA-TG)_n (P4), and non-responsive copolymer mPEG-b-PCL (P5) were constructed. pH/ROS-responsive properties were characterized by proton nuclear magnetic resonance (¹H NMR) and dynamic light scattering (DLS). Anticancer chemotherapeutic agent gemcitabine (GEM) or fluorescent substance Nile Red (NR) were loaded in the polymer micelles. Results of the mitochondrial colocalization experiment indicate that (5-carboxypentyl)(triphenyl)phosphonium bromide (TPP)-functionalized P1 micelles could be efficiently targeted and located in mitochondria. Results of the cellular uptake experiment showed that pH/ROS dual-responsive GEM-loaded P1 and P2 micelles have faster internalized and entry nucleus rates than single-responsive or non-responsive GEM-loaded micelles. The in vitro release experiment suggests pH/ROS dual-responsive GEM/P1 and GEM/P2 micelles have higher cumulative release than single-responsive GEM/P3 and GEM/P4 micelles. The in vitro cytotoxic experiment shows that the mitochondria-targeted dual-responsive GEM/P1 micelles had the lowest IC50 values, and the cytotoxic effect of dual-responsive GEM/P2 micelles was superior to the single-responsive and non-responsive drug-loaded micelles.     

The influence of linoleic acid intake on the kinetics of adenine nucleotide translocase

Dietary enrichment of mitochondrial polyenoic fatty acid content was associated with increased respiratory activity (Abuirmeileh and Elson). The influence of the membrane lipid composition on the adenine nucleotide translocase (AdNT) was studied in rats that were fed diets formulated with beef tallow (BT) to provide low or safflower oil (SO) to provide high contents of linoleic acid. The phosphatidylcholine/phosphatidylethanolamine ratio was 40% higher in the SO mitochondria due primarily to an increase in phosphatidylcholine. SO mitochondria exhibited 25% higher state 3 respiration, 50% higher state 4 respiration and 13% higher net ADP-dependent respiration than did the BT mitochondria. The relative RCR and ADP/O values of the SO mitochondria were slightly but significantly (p<0.01) lower. The kinetics of the AdNT were determined by the back exchange method (Pfaff and Klingenberg). At all assay temperatures, SO mitochondria exhibited a higher K_m for ADP. However, addition of 5 mM carnitine to the assay mixture increased the affinity of the SO-AdNT giving K_m values similar to that of the BT-AdNT. Washing the mitochondria with fat-free BSA had a similar, but lesser, effect. At 25 C and 37 C, the V_max of the SO-AdNT were increased by 11–15% (p<0.05) which was independent of either BSA-wash or the presence of carnitine. According to Dixon plot studies, the SO-AdNT had a comparable K_i for atractylate but a slightly lower K_i for palmitoyl-CoA inhibition. The accumulation of acyl-CoA esters in the SO mitochondria was not ruled out. The overall results suggest that changes in mitochondrial membrane lipids accommodated an increased V_max of the SO-AdNT, which in turn accounted for that part of the increased state 3 respiration dependent on ADP translocation.     

Transport Pathways—Proton Motive Force Interrelationship in Durum Wheat Mitochondria

In durum wheat mitochondria (DWM) the ATP-inhibited plant mitochondrial potassium channel (PmitoK_ATP) and the plant uncoupling protein (PUCP) are able to strongly reduce the proton motive force (pmf) to control mitochondrial production of reactive oxygen species; under these conditions, mitochondrial carriers lack the driving force for transport and should be inactive. However, unexpectedly, DWM uncoupling by PmitoK_ATP neither impairs the exchange of ADP for ATP nor blocks the inward transport of Pi and succinate. This uptake may occur via the plant inner membrane anion channel (PIMAC), which is physiologically inhibited by membrane potential, but unlocks its activity in de-energized mitochondria. Probably, cooperation between PIMAC and carriers may accomplish metabolite movement across the inner membrane under both energized and de-energized conditions. PIMAC may also cooperate with PmitoK_ATP to transport ammonium salts in DWM. Interestingly, this finding may trouble classical interpretation of in vitro mitochondrial swelling; instead of free passage of ammonia through the inner membrane and proton symport with Pi, that trigger metabolite movements via carriers, transport of ammonium via PmitoK_ATP and that of the counteranion via PIMAC may occur. Here, we review properties, modulation and function of the above reported DWM channels and carriers to shed new light on the control that they exert on pmf and vice-versa.     

The Role of Mitochondria Dysfunction in Inflammatory Bowel Diseases and Colorectal Cancer

Inflammatory bowel disease (IBD) is one of the leading gut chronic inflammation disorders, especially prevalent in Western countries. Recent research suggests that mitochondria play a crucial role in IBD development and progression to the more severe disease—colorectal cancer (CRC). In this review, we focus on the role of mitochondrial mutations and dysfunctions in IBD and CRC. In addition, main mitochondria-related molecular pathways involved in IBD to CRC transition are discussed. Additionally, recent publications dedicated to mitochondria-targeted therapeutic approaches to cure IBD and prevent CRC progression are discussed.     

Dual Inhibition of Pyruvate Dehydrogenase Complex and Respiratory Chain Complex Induces Apoptosis by a Mitochondria-Targeted Fluorescent Organic Arsenical in vitro and in vivo

Based on the potential therapeutic value in targeting mitochondria and the fluorophore tracing ability, a fluorescent mitochondria-targeted organic arsenical PDT-PAO-F16 was fabricated, which not only visualized the cellular distribution, but also exerted anti-cancer activity in vitro and in vivo via targeting pyruvate dehydrogenase complex (PDHC) and respiratory chain complexes in mitochondria. In details, PDT-PAO-F16 mainly accumulated into mitochondria within hours and suppressed the activity of PDHC resulting in the inhibition of ATP synthesis and thermogenesis disorder. Moreover, the suppression of respiratory chain complex I and IV accelerated the mitochondrial dysfunction leading to caspase family-dependent apoptosis. In vivo, the acute promyelocytic leukemia was greatly alleviated in the PDT-PAO-F16 treated group in APL mice model. Our results demonstrated the organic arsenical precursor with fluorescence imaging and target-anticancer efficacy is a promising anticancer drug.     

Radiolabelled CCK2R Antagonists Containing PEG Linkers: Design,Synthesis and Evaluation

The cholecystokinin-2/gastrin receptor (CCK₂R) is considered a suitable target for the development of radiolabelled antagonists, due to its overexpression in various tumours, but no such compounds are available in clinical use. Therefore, we designed novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated ligands based on CCK₂R antagonist Z360/nastorazepide. As a proof of concept that CCK₂R antagonistic activity can be retained by extending the Z360/nastorazepide structure using suitable linker, we present herein three compounds containing various PEG linkers synthesised on solid phase and in solution. The antagonistic properties were measured in a functional assay in the A431-CCK₂R cell line (in the presence of agonist G17), with IC₅₀ values of 3.31, 4.11 and 10.4 nM for compounds containing PEG₄, PEG₆ and PEG₁₂, respectively. All compounds were successfully radiolabelled with indium-111, lutetium-177 and gallium-68 (incorporation of radiometal >95 %). The gallium-68-labelled compounds were stable for up to 2 h (PBS, 37 °C). log D_7.4 values were determined for indium-111- and gallium-68-labelled compounds, showing improved hydrophilicity compared to the reference compound.     

A NIR Emitting Cyanine with Large Stokes’ Shift for Mitochondria and Identification of their Membrane Potential Disruption

An NIR emitting (λ_em≈730 nm) cyanine probe ExCy was synthesized in good yields by extending the π-conjugation length (i. e., with furan moiety) to the donor-accepter system. ExCy exhibited a large Stokes’ shift (Δλ≈100 nm) due to strong intramolecular charge transfer (ICT), and high fluorescence quantum yield (Φ_fl≈0.47 in DCM). Due to its low fluorescence in an aqueous environment (Φ_fl≈0.007 in H₂O), the probe exhibited the potential of achieving a large fluorescence turn-on upon entering a hydrophobic cellular environment. Fluorescence confocal microscopy studies revealed that ExCy was readily excitable with a far-red laser line (i. e., 640 nm) while the corresponding emission was collected in the NIR region. ExCy exhibited excellent selectivity towards live cell mitochondria according to the co-localization studies. The probe also exhibited high photostability, long-term imaging ability and wash-free staining ability, when being applied to live cells. Our studies indicated that the mitochondrial localization of ExCy was dependent on the membrane potential of the mitochondria. ExCy was successfully utilized as a mitochondrial membrane potential dysfunction indicator to visually identify cells with mitochondrial dysfunction via fluorescence confocal microscopy. ExCy was further examined for potential in vivo imaging of zebrafish.     

Targeting mitochondria

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are closely linked to degenerative diseases such as Alzheimer's disease, Parkinson's, neuronal death including ischemic and hemorrhagic stroke, acute and chronic degenerative cardiac myocyte death, and cancer. As a byproduct of oxidative phosphorylation, a steady stream of reactive species emerge from our cellular energy plants, the mitochondria. ROS and RNS potentially cause damage to all cellular components. Structure alteration, biomolecule fragmentation, and oxidation of side chains are trade-offs of cellular energy production. ROS and RNS escape results in the activation of cytosolic stress pathways, DNA damage, and the upregulation of JNK, p38, and p53. Incomplete scavenging of ROS and RNS particularly affects the mitochondrial lipid cardiolipin (CL), triggers the release of mitochondrial cytochrome c, and activates the intrinsic death pathway. Due to the active redox environment and the excess of NADH and ATP at the inner mitochondrial membrane, a broad range of agents including electron acceptors, electron donors, and hydride acceptors can be used to influence the biochemical pathways. The key to therapeutic value is to enrich selective redox modulators at the target sites. Our approach is based on conjugating nitroxides to segments of natural products with relatively high affinity for mitochondrial membranes. For example, a modified gramicidin S segment was successfully used for this purpose and proven to be effective in preventing superoxide production in cells and CL oxidation in mitochondria and in protecting cells against a range of pro-apoptotic triggers such as actinomycin D, radiation, and staurosporine. More importantly, these mitochondria-targeted nitroxide/gramicidin conjugates were able to protect against apoptosis in vivo by preventing CL oxidation induced by intestinal hemorrhagic shock. Optimization of nitroxide carriers could lead to a new generation of effective antiapoptotic agents acting at an early mitochondrial stage. Alternative chemistry-based approaches to targeting mitochondria include the use of proteins and peptides, as well as the attachment of payloads to lipophilic cationic compounds, sulfonylureas, anthracyclines, and other agents with proven or hypothetical affinities for mitochondria. Manganese superoxide dismutase (MnSOD), SS tetrapeptides with 2',6'-dimethyltyrosine (Dmt) residues, rhodamine, triphenylphosphonium salts, nonopioid analgesics, adriamycin, and diverse electron-rich aromatics and stilbenes were used to influence mitochondrial biochemistry and the biology of aging. Some general structural principles for effective therapeutic agents are now emerging. Among these are the presence of basic or positively charged functional groups, hydrophobic substructures, and, most promising for future selective strategies, classes of compounds that are actively shuttled into mitochondria, bind to mitochondria-specific proteins, or show preferential affinity to mitochondria-specific lipids.     

Bee Venom Melittin Disintegrates the Respiration of Mitochondria in Healthy Cells and Lymphoblasts,and Induces the Formation of Non-Bilayer Structures in Model Inner Mitochondrial Membranes

In this paper, we examined the effects of melittin, a bee venom membrane-active peptide, on mitochondrial respiration and cell viability of healthy human lymphocytes (HHL) and Jurkat cells, as well as on lymphoblasts from acute human T cell leukemia. The viability of melittin-treated cells was related to changes in O₂ consumption and in the respiratory control index (RCI) of mitochondria isolated from melittin-pretreated cells as well as of mitochondria first isolated from cells and then directly treated with melittin. It was shown that melittin is three times more cytotoxic to Jurkat cells than to HHL, but O₂ consumption and RCI values of mitochondria from both cell types were equally affected by melittin when melittin was directly added to mitochondria. To elucidate the molecular mechanism of melittin’s cytotoxicity to healthy and cancer cells, the effects of melittin on lipid-packing and on the dynamics in model plasma membranes of healthy and cancer cells, as well as of the inner mitochondrial membrane, were studied by EPR spin probes. The affinity of melittin binding to phosphatidylcholine, phosphatidylserine, phosphatidic acid and cardiolipin, and binding sites of phospholipids on the surface of melittin were studied by ³¹P-NMR, native PAGE and AutoDock modeling. It is suggested that the melittin-induced decline of mitochondrial bioenergetics contributes primarily to cell death; the higher cytotoxicity of melittin to cancer cells is attributed to its increased permeability through the plasma membrane.     

Effect of Quercetin on mitoBKCa Channel and Mitochondrial Function in Human Bronchial Epithelial Cells Exposed to Particulate Matter

Particulate matter (PM) exposure increases reactive oxygen species (ROS) levels. It can lead to inflammatory responses and damage of the mitochondria thus inducing cell death. Recently, it has been shown that potassium channels (mitoK) located in the inner mitochondrial membrane are involved in cytoprotection, and one of the mechanisms involves ROS. To verify the cytoprotective role of mitoBK_Ca, we performed a series of experiments using a patch-clamp, transepithelial electrical resistance assessment (TEER), mitochondrial respiration measurements, fluorescence methods for the ROS level and mitochondrial membrane potential assessment, and cell viability measurements. In the human bronchial epithelial cell model (16HBE14σ), PM < 4 μm in diameter (SRM-PM4.0) was used. We observed that PM decreased TEER of HBE cell monolayers. The effect was partially abolished by quercetin, a mitoBK_Ca opener. Consequently, quercetin decreased the mitochondrial membrane potential and increased mitochondrial respiration. The reduction of PM-induced ROS level occurs both on cellular and mitochondrial level. Additionally, quercetin restores HBE cell viability after PM administration. The incubation of cells with PM substantially reduced the mitochondrial function. Isorhamnetin had no effect on TEER, the mitoBK_Ca activity, respiratory rate, or mitochondrial membrane potential. Obtained results indicate that PM has an adverse effect on HBE cells at the cellular and mitochondrial level. Quercetin is able to limit the deleterious effect of PM on barrier function of airway epithelial cells. We show that the effect in HBE cells involves mitoBK_Ca channel-activation. However, quercetin’s mechanism of action is not exclusively determined by modulation of the channel activity.     

Effect of docosahexaenoic acid on mouse mitochondrial membrane properties

Long-chain polyunsaturated (n-3) fatty acids have been proposed to be involved in a wide variety of biological activities. In this study, mitochondrial docosahexaenoic acid (DHA) levels were increased by either dietary manipulation or by fusing the mitochondria with phospholipid vesicles made from 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0/22:6 PC). The fused mitochondria exhibited a DHA-induced decrease in respiratory control index (RCI) and membrane potential and an increase in proton movement. The modified mitochondria also demonstrated an increase in fluidity (as detected by 1,6-diphenyl-1,3,5-hexatriene anisotropy) and changes in membrane structure detected by the fluorescence probes MC540 and pyrene decanoate. Proton movement in lipid vesicles made from mitochondrial lipid extracts was shown to be enhanced by incorporated 18:0/22:6 PC. Mitochondria were isolated from young (5-mon) and old (24-mon) mice which were maintained on either a diet rich in saturated fats (hydrogenated coconut oil) or rich in n−3 polyunsaturated fats (menhaden oil). Mitochondrial bioenergetic function was followed by RCI, state 3 respiration, ATP level, and phosphate uptake. In addition, lipid composition, phospholipid area/molecule, and extent of lipid peroxidation were also determined. Decreases in RCI for the menhaden oil diet-modified mitochondria paralleled those in which DHA levels were enhanced by fusion with phospholipid vesicles. RCI reductions are attributed to DHA-induced increases in H⁺ movement, producing diminished mitochondrial membrane potentials. One purpose of this project was to determine if the deleterious effects of aging on mitochondrial bioenergetic function could be reversed by addition of n−3 fatty acids. The experiments reported here indicate that incorporation of long-chain polyunsaturated n−3 fatty acids into mitochondrial membranes does not appear likely to reverse the effects of age on mitochondrial function.