Lipid Peroxidation-derived Aldehydes Research Articles

Oxidative Stress-mediated Lipid Peroxidation-derived Lipid Aldehydes in the Pathophysiology of Neurodegenerative Diseases.

Neurodegenerative diseases such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis cause damage and gradual loss of neurons affecting the central nervous system. Neurodegenerative diseases are most commonly seen in the ageing process. Ageing causes increased reactive oxygen species and decreased mitochondrial ATP generation, resulting in redox imbalance and oxidative stress. Oxidative stress-generated free radicals cause damage to membrane lipids containing polyunsaturated fatty acids, leading to the formation of toxic lipid aldehyde products such as 4- hydroxynonenal and malondialdehyde. Several studies have shown that lipid peroxidation-derived aldehyde products form adducts with cellular proteins, altering their structure and function. Thus, these lipid aldehydes could act as secondary signaling intermediates, modifying important metabolic pathways, and contributing to the pathophysiology of several human diseases, including neurodegenerative disorders. Additionally, they could serve as biomarkers for disease progression. This narrative review article discusses the biological and clinical significance of oxidative stress-mediated lipid peroxidation-derived lipid aldehydes in the pathophysiology of various neurodegenerative diseases.

Skeletal muscle analysis of cancer patients reveals a potential role for carnosine in muscle wasting.

Muscle wasting during cancer cachexia is mediated by protein degradation via autophagy and ubiquitin-linked proteolysis. These processes are sensitive to changes in intracellular pH ([pH]i ) and reactive oxygen species, which in skeletal muscle are partly regulated by histidyl dipeptides, such as carnosine. These dipeptides, synthesized by the enzyme carnosine synthase (CARNS), remove lipid peroxidation-derived aldehydes, and buffer [pH]i . Nevertheless, their role in muscle wasting has not been studied. Histidyl dipeptides in the rectus abdominis (RA) muscle and red blood cells (RBCs) of male and female controls (n=37), weight stable (WS: n=35), and weight losing (WL; n=30) upper gastrointestinal cancer (UGIC) patients, were profiled by LC-MS/MS. Expression of enzymes and amino acid transporters, involved in carnosine homeostasis, was measured by Western blotting and RT-PCR. Skeletal muscle myotubes were treated with Lewis lung carcinoma conditioned medium (LLC CM), and β-alanine to study the effects of enhancing carnosine production on muscle wasting. Carnosine was the predominant dipeptide present in the RA muscle. In controls, carnosine levels were higher in men (7.87±1.98nmol/mg tissue) compared with women (4.73±1.26nmol/mg tissue; P=0.002). In men, carnosine was significantly reduced in both the WS (5.92±2.04nmol/mg tissue, P=0.009) and WL (6.15±1.90nmol/mg tissue; P=0.030) UGIC patients, compared with controls. In women, carnosine was decreased in the WL UGIC (3.42±1.33nmol/mg tissue; P=0.050), compared with WS UGIC patients (4.58±1.57nmol/mg tissue), and controls (P=0.025). Carnosine was significantly reduced in the combined WL UGIC patients (5.12±2.15nmol/mg tissue) compared with controls (6.21±2.24nmol/mg tissue; P=0.045). Carnosine was also significantly reduced in the RBCs of WL UGIC patients (0.32±0.24pmol/mg protein), compared with controls (0.49±0.31pmol/mg protein, P=0.037) and WS UGIC patients (0.51±0.40pmol/mg protein, P=0.042). Depletion of carnosine diminished the aldehyde-removing ability in the muscle of WL UGIC patients. Carnosine levels were positively associated with decreases in skeletal muscle index in the WL UGIC patients. CARNS expression was decreased in the muscle of WL UGIC patients and myotubes treated with LLC-CM. Treatment with β-alanine, a carnosine precursor, enhanced endogenous carnosine production and decreased ubiquitin-linked protein degradation in LLC-CM treated myotubes. Depletion of carnosine could contribute to muscle wasting in cancer patients by lowering the aldehyde quenching abilities. Synthesis of carnosine by CARNS in myotubes is particularly affected by tumour derived factors and could contribute to carnosine depletion in WL UGIC patients. Increasing carnosine in skeletal muscle may be an effective therapeutic intervention to prevent muscle wasting in cancer patients.

Massive foreign body reaction and osteolysis following primary anterior cruciate ligament reconstruction with the ligament augmentation and reconstruction system (LARS): a case report with histopathological and physicochemical analysis

BackgroundAutologous hamstrings and patellar tendon have historically been considered the gold standard grafts for anterior cruciate ligament reconstruction (ACLR). In the last decades, the utilization of synthetic grafts has re-emerged due to advantageous lack of donor site morbidity and more rapid return to sport. The Ligament Augmentation and Reconstruction System (LARS) has demonstrated to be a valid and safe option for ACLR in the short term. However, recent studies have pointed out the notable frequency of associated complications, including synovitis, mechanical failure, and even chondrolysis requiring joint replacement.Case presentationWe report the case of a 23-year-old male who developed a serious foreign body reaction with wide osteolysis of both femoral and tibial tunnels following ACLR with LARS. During first-stage arthroscopy, we performed a debridement of the pseudocystic mass incorporating the anterior cruciate ligament (ACL) and extending towards the tunnels, which were filled with autologous anterior iliac crest bone graft chips. Histological analysis revealed the presence of chronic inflammation, fibrosis, and foreign body giant cells with synthetic fiber inclusions. Furthermore, physicochemical analysis showed signs of fiber depolymerization, increased crystallinity and formation of lipid peroxidation-derived aldehydes, which indicate mechanical aging and instability of the graft. After 8 months, revision surgery was performed and ACL revision surgery with autologous hamstrings was successfully carried out.ConclusionsThe use of the LARS grafts for ACLR should be cautiously contemplated considering the high risk of complications and early failure.

Targeted delivery of acrolein scavenger hydralazine in spinal cord injury using folate-linker-drug conjugation

Oxidative stress has been shown to play a critical pathogenic role in functional loss after spinal cord injury (SCI). As a direct result of oxidative stress, lipid peroxidation-derived aldehydes have emerged as key culprits that sustain secondary injury and contribute significantly to pathological outcomes. Acrolein, a neurotoxin, has been shown to be elevated in SCI and can result in post-SCI neurological deficits. Reducing acrolein has therefore emerged as a novel and effective therapeutic strategy in SCI. Previous studies have revealed that hydralazine, an FDA approved blood pressure lowering medication, when administered after SCI shows strong acrolein scavenging capabilities and significantly improves cellular and behavioral outcomes. However, while effective at scavenging acrolein, hydralazine's blood pressure lowering activity can have a detrimental impact on neurotrauma patients. Here, our goal was to preserve the acrolein scavenging capability while mitigating the effect of hydralazine on blood pressure. We accomplished this using a folate-targeted delivery system to deploy hydralazine to the folate receptor positive inflammatory site of the cord injury. Using a model of rat SCI, we found that this system is effective for targeting the injury site, and that folate targeted hydralazine can scavenge acrolein without significantly impacting blood pressure.

Short-chain aldehydes increase aluminum retention and sensitivity by enhancing cell wall polysaccharide contents and pectin demethylation in wheat seedlings

Upon environmental stimuli, aldehydes are generated downstream of reactive oxygen species and thereby contribute to severe cell damage. In this study, using two wheat genotypes differing in aluminum (Al) tolerance, we investigated the effects of lipid peroxidation-derived aldehydes on cell wall composition and subsequent Al-binding capacities. The spatial accumulation of Al along wheat roots was found to the generation of reactive aldehydes, which are highly localized to the apical regions of roots. Elimination of aldehydes by carnosine significantly reduced Al contents in root tips, with a concomitant alleviation of root growth inhibition. In contrast, root growth and Al accumulation were exacerbated by application of the short-chain aldehyde (E)−2-hexenal. We further confirmed that cell wall binding capacity, rather than malate efflux or pH alteration strategies, is associated with the aldehyde-induced accumulation of Al. Scavenging of lipid-derived aldehydes reduced Al accumulation in the pectin and hemicellulose 1 (HC1) fractions of root cell walls, whereas exposure to (E)−2-hexenal promoted a further accumulation of Al, particularly in the cell wall HC1 fraction of the Al-sensitive genotype. Different strategies were introduced by pectin and HC1 to accumulate Al in response to aldehydes in wheat roots. Accumulation in pectin is based on a reduction of methylation levels in response to elevated pectin methylesterase activity and gene expression, whereas that in HC1 is associated with an increase in polysaccharide contents. These findings indicate that aldehydes exacerbate Al phytotoxicity by enhancing Al retention in cell wall polysaccharides.

Critical role of mitochondrial aldehyde dehydrogenase 2 in acrolein sequestering in rat spinal cord injury.

Lipid peroxidation-derived aldehydes, such as acrolein, the most reactive aldehyde, have emerged as key culprits in sustaining post-spinal cord injury (SCI) secondary pathologies leading to functional loss. Strong evidence suggests that mitochondrial aldehyde dehydrogenase-2 (ALDH2), a key oxidoreductase and powerful endogenous anti-aldehyde machinery, is likely important for protecting neurons from aldehydes-mediated degeneration. Using a rat model of spinal cord contusion injury and recently discovered ALDH2 activator (Alda-1), we planned to validate the aldehyde-clearing and neuroprotective role of ALDH2. Over an acute 2 day period post injury, we found that ALDH2 expression was significantly lowered post-SCI, but not so in rats given Alda-1. This lower enzymatic expression may be linked to heightened acrolein-ALDH2 adduction, which was revealed in co-immunoprecipitation experiments. We have also found that administration of Alda-1 to SCI rats significantly lowered acrolein in the spinal cord, and reduced cyst pathology. In addition, Alda-1 treatment also resulted in significant improvement of motor function and attenuated post-SCI mechanical hypersensitivity up to 28 days post-SCI. Finally, ALDH2 was found to play a critical role in in vitro protection of PC12 cells from acrolein exposure. It is expected that the outcome of this study will broaden and enhance anti-aldehyde strategies in combating post-SCI neurodegeneration and potentially bring treatment to millions of SCI victims. All animal work was approved by Purdue Animal Care and Use Committee (approval No. 1111000095) on January 1, 2021.

Physiological and Pathological Roles of Aldose Reductase.

Aldose reductase (AR) is an aldo-keto reductase that catalyzes the first step in the polyol pathway which converts glucose to sorbitol. Under normal glucose homeostasis the pathway represents a minor route of glucose metabolism that operates in parallel with glycolysis. However, during hyperglycemia the flux of glucose via the polyol pathway increases significantly, leading to excessive formation of sorbitol. The polyol pathway-driven accumulation of osmotically active sorbitol has been implicated in the development of secondary diabetic complications such as retinopathy, nephropathy, and neuropathy. Based on the notion that inhibition of AR could prevent these complications a range of AR inhibitors have been developed and tested; however, their clinical efficacy has been found to be marginal at best. Moreover, recent work has shown that AR participates in the detoxification of aldehydes that are derived from lipid peroxidation and their glutathione conjugates. Although in some contexts this antioxidant function of AR helps protect against tissue injury and dysfunction, the metabolic transformation of the glutathione conjugates of lipid peroxidation-derived aldehydes could also lead to the generation of reactive metabolites that can stimulate mitogenic or inflammatory signaling events. Thus, inhibition of AR could have both salutary and injurious outcomes. Nevertheless, accumulating evidence suggests that inhibition of AR could modify the effects of cardiovascular disease, asthma, neuropathy, sepsis, and cancer; therefore, additional work is required to selectively target AR inhibitors to specific disease states. Despite past challenges, we opine that a more gainful consideration of therapeutic modulation of AR activity awaits clearer identification of the specific role(s) of the AR enzyme in health and disease.

Probiotics-Based Treatment as an Integral Approach for Alcohol Use Disorder in Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is one of the leading causes of morbidity among adults with alcohol use disorder (AUD) worldwide. Its clinical course ranges from steatosis to alcoholic hepatitis, progressing to more severe forms of liver damage, such as cirrhosis and hepatocellular carcinoma. The pathogenesis of ALD is complex and diverse elements are involved in its development, including environmental factors, genetic predisposition, the immune response, and the gut-liver axis interaction. Chronic alcohol consumption induces changes in gut microbiota that are associated with a loss of intestinal barrier function and inflammatory responses which reinforce a liver damage progression triggered by alcohol. Alcohol metabolites such as acetaldehyde, lipid peroxidation-derived aldehyde malondialdehyde (MDA), and protein-adducts act as liver-damaging hepatotoxins and potentiate systemic inflammation. Additionally, ethanol causes direct damage to the central nervous system (CNS) by crossing the blood-brain barrier (BBB), provoking oxidative stress contributing to neuroinflammation. Overall, these processes have been associated with susceptibility to depression, anxiety, and alcohol craving in ALD. Recent evidence has shown that probiotics can reverse alcohol-induced changes of the microbiota and prevent ALD progression by restoring gut microbial composition. However, the impact of probiotics on alcohol consumption behavior has been less explored. Probiotics have been used to treat various conditions by restoring microbiota and decreasing systemic and CNS inflammation. The results of some studies suggest that probiotics might improve mental function in Alzheimer’s, autism spectrum disorder, and attenuated morphine analgesic tolerance. In this sense, it has been observed that gut microbiota composition alterations, as well as its modulation using probiotics, elicit changes in neurotransmitter signals in the brain, especially in the dopamine reward circuit. Consequently, it is not difficult to imagine that a probiotics-based complementary treatment to ALD might reduce disease progression mediated by lower alcohol consumption. This review aims to present an update of the pathophysiologic mechanism underlying the microbiota-gut-liver-brain axis in ALD, as well as to provide evidence supporting probiotic use as a complementary therapy to address alcohol consumption disorder and its consequences on liver damage.

4-Hydroxynonenal Contributes to Fibroblast Senescence in Skin Photoaging Evoked by UV-A Radiation

Solar ultraviolet A (UV-A) radiation promotes a huge variety of damages on connective tissues and dermal fibroblasts, including cellular senescence, a major contributor of skin photoaging. The mechanisms of skin photoaging evoked by UV-A partly involve the generation of reactive oxygen species and lipid peroxidation. We previously reported that 4-hydroxynonenal (HNE), a lipid peroxidation-derived aldehyde, forms adducts on elastin in the skins of UV-A irradiated hairless mice, possibly contributing to actinic elastosis. In the present study, we investigated whether and how HNE promotes fibroblast senescence in skin photoaging. Dermal fibroblasts of skins from UV-A-exposed hairless mice exhibited an increased number of γH2AX foci characteristic of cell senescence, together with an accumulation of HNE adducts partly colocalizing with the cytoskeletal protein vimentin. Murine fibroblasts exposed to UV-A radiation (two cycles of 15 J/cm2), or HNE (30 µM, 4 h), exhibited senescence patterns characterized by an increased γH2AX foci expression, an accumulation of acetylated proteins, and a decreased expression of the sirtuin SIRT1. HNE adducts were detected on vimentin in cultured fibroblasts irradiated by UV-A or incubated with HNE. The HNE scavenger carnosine prevented both vimentin modification and fibroblast senescence evoked by HNE in vitro and in the skins of UV-A-exposed mice. Altogether, these data emphasize the role of HNE and lipid peroxidation-derived aldehydes in fibroblast senescence, and confirm the protective effect of carnosine in skin photoaging.

A role for 4-hydroxy-2-nonenal in premature placental senescence in preeclampsia and intrauterine growth restriction

Premature placental senescence is a hallmark of pregnancy-related disorders such as intrauterine growth restriction (IUGR) and preeclampsia (PE), two major cause of maternal and neonatal morbidity and mortality. Oxidative stress and lipid peroxidation are involved in the pathogenesis of PE and IUGR, and may play a role in placental aging. In this study, we investigated whether 4-hydroxy-2-nonenal (HNE), a lipid peroxidation-derived aldehyde present in preeclamptic placentas, may contribute to premature senescence in placenta-related complications.Placentas from PE-affected women, exhibited several senescence patterns, such as an increased expression of phosphorylated (serine-139) histone γH2AX, a sensitive marker of double-stranded DNA breaks, the presence of lipofuscin granules, and an accumulation of high molecular weight cross-linked and ubiquitinated proteins. PE placentas showed an accumulation of acetylated proteins consistent with the presence of HNE-adducts on sirtuin 1 (SIRT1). Likewise, oxidative stress and senescence markers together with SIRT1 modification by HNE, were observed in murine placentas from mice treated with lipopolysaccharide during gestation and used as models of IUGR. The addition of HNE and ONE (4-oxo-2-nonenal), to cultured HTR-8/SVneo human trophoblasts activated the senescence-associated- β-galactosidase, and generated an accumulation of acetylated proteins, consistent with a modification of SIRT1 by HNE.Altogether, these data emphasize the role of HNE and lipid peroxidation-derived aldehydes in premature placental senescence in PE and IUGR, and more generally in pathological pregnancies.

Trans,trans-2,4-Decadienal induces endothelial cell injury by impairing mitochondrial function and autophagic flux.

This study investigated the toxic effects of trans,trans-2,4-decadienal (tt-DDE) on vascular endothelial cells as well as the underlying mechanisms involved. Human umbilical vascular endothelial cells (HUVECs) were treated with different concentrations of tt-DDE for 24 h, and cell viability, colony formation ability, apoptosis, mitochondrial function and autophagy pathway were determined. The results showed that tt-DDE dose-dependently inhibited cell viability and colony formation, and increased lactate dehydrogenase (LDH) release and apoptosis in HUVECs. Besides, tt-DDE exposure induced extensive mitochondrial damage, as evidenced by the decreased mitochondrial DNA copy number, ATP synthesis, and mitochondrial membrane potential, and increased mitochondrial reactive oxygen species (ROS) production and cytochrome c release from mitochondria. tt-DDE also induced mitochondrial fragmentation and fission by increasing DNM1L protein expression and DNM1L mitochondrial translocation. Additionally, tt-DDE treatment resulted in the blockage of autophagic flux and accumulation of autophagosomes in endothelial cells. Further investigation revealed that the inhibition of autophagy by 3-methyladenine aggravated tt-DDE-induced mitochondrial dysfunction and cell injury. However, scavenging of ROS by N-acetyl-l-cysteine (NAC) significantly prevented tt-DDE-induced mitochondrial damage, autophagy dysfunction, and cell injury. These data indicated that tt-DDE induced endothelial cell injury through impairing mitochondrial function and autophagic flux.

4-Hydroxy-Trans-2-Nonenal in the Regulation of Anti-Oxidative and Pro-Inflammatory Signaling Pathways.

Recent studies indicate that 4-hydroxy-trans-2-nonenal (HNE), a major oxidative stress triggered lipid peroxidation-derived aldehyde, plays a critical role in the pathophysiology of various human pathologies including metabolic syndrome, diabetes, cardiovascular, neurological, immunological, and age-related diseases and various types of cancer. HNE is the most abundant and toxic α, β-unsaturated aldehyde formed during the peroxidation of polyunsaturated fatty acids in a series of free radical-mediated reactions. The presence of an aldehyde group at C1, a double bond between C2 and C3 and a hydroxyl group at C4 makes HNE a highly reactive molecule. These strong reactive electrophilic groups favor the formation of HNE adducts with cellular macromolecules such as proteins and nucleic acids leading to the regulation of various cell signaling pathways and processes involved in cell proliferation, differentiation, and apoptosis. Many studies suggest that the cell-specific intracellular concentrations of HNE dictate the anti-oxidative and pro-inflammatory activities of this important molecule. In this review, we focused on how HNE could alter multiple anti-oxidative defense pathways and pro-inflammatory cytotoxic pathways by interacting with various cell-signaling intermediates.

Caffeic acid phenethyl ester potentiates gastric cancer cell sensitivity to doxorubicin and cisplatin by decreasing proteasome function

Caffeic acid phenethyl ester (CAPE) is a major propolis component that possesses a variety of pharmacological properties such as antioxidant and anticancer effects. Herein, we investigated the effectiveness of CAPE on cytotoxicity of clinically used anticancer drugs, doxorubicin (DXR) and cisplatin (CDDP), in parental and the drug-resistant cells of stomach (MKN45) and colon (LoVo) cancers. Concomitant treatment with CAPE potentiated apoptotic effects of DXR and CDDP against the parental cells. The treatment significantly reduced the production of reactive oxygen species elicited by DXR but did not affect the DXR-mediated accumulation of 4-hydroxy-2-nonenal, a lipid peroxidation-derived aldehyde. Intriguingly, treatment of parental MKN45 cells with CAPE alone reduced 26S proteasome-based proteolytic activities, in which a chymotrypsin-like activity was most affected. This effect of CAPE was the most prominent among those of eight flavonoids and nine cinnamic acid derivatives and was also observed in parental LoVo cells. In the DXR-resistant or CDDP-resistant cells, the chymotrypsin-like activity was highly up-regulated and significantly decreased by CAPE treatment, which sensitized the resistant cells to DXR and CDDP. Reverse transcription-PCR analysis showed that CAPE treatment led to downregulation of five proteasome subunits (PSMB1-PSMB5) and three immunoproteasome subunits (PSMB8-PSMB10) in DXR-resistant MKN45 cells. The results suggest that CAPE enhances sensitivity of these cancer cells and their chemoresistant cells to DXR and CDDP, most notably through decreasing proteasome function. Thus, CAPE may be valuable as an adjuvant for DXR or CDDP chemotherapy in gastric cancer.

Effect of the Lipid Peroxidation Product 4-Hydroxynonenal on Neuroinflammation in Microglial Cells: Protective Role of Quercetin and Monochloropivaloylquercetin

The lipid peroxidation-derived aldehyde 4-hydroxynonenal (HNE) has been implicated in a number of oxidative stress-induced inflammatory pathologies such as neurodegenerative diseases and aging. In this regard, we investigated the effects of HNE on neuroinflammatory responses by measuring cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) induction with cytokine production. In addition, we measured nuclear factor erythroid 2-related factor 2 (Nrf-2)/Kelch-like ECH-associated protein 1 (Keap1) signaling proteins, and antioxidant enzymes heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate dehydrogenase, quinone 1 (NQO1), and compared the results with quercetin and monochloropivaloylquercetin (MPQ) pretreated microglial cells. Cytotoxicity was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and production of cytokines was determined by cytokine array. Furthermore, intracellular Nfr2/Keap1 signaling proteins, HO-1, NQO1, and COX-2 expression were analyzed by western blot in 2.5 μM HNE treated BV-2 cells. Inducible nitric oxide synthase (iNOS) and COX-2 mRNA levels were measured with reverse transcription-quantitative polymerase chain reaction. HNE induced both COX-2 mRNA and protein levels, iNOS mRNA expression, and cytokine production. In addition, HNE markedly increased Keap1 levels and decreased cytoplasmic Nrf-2 expression with antioxidant enzyme HO-1 levels. Quercetin and monochloropivaloylquercetin treatment alleviated neuroinflammatory responses in microglial cells, by decreasing COX-2 mRNA expression. Monochloropivaloylquercetin decreased cytoplasmic Keap1 levels and increased nuclear translocation of Nrf-2 resulted in induction of HO-1 and NQO1 expression. These results suggest that HNE could be a link between oxidative stress and inflammation in BV-2 microglia cells. In particular, monochloropivaloylquercetin alleviated inflammation, probably by decreasing the expression of proinflammatory genes and strengthening the antioxidant defense system.

Lipid Peroxidation-Derived Aldehydes, 4-Hydroxynonenal and Malondialdehyde in Aging-Related Disorders.

Among the various mechanisms involved in aging, it was proposed long ago that a prominent role is played by oxidative stress. A major way by which the latter can provoke structural damage to biological macromolecules, such as DNA, lipids, and proteins, is by fueling the peroxidation of membrane lipids, leading to the production of several reactive aldehydes. Lipid peroxidation-derived aldehydes can not only modify biological macromolecules, by forming covalent electrophilic addition products with them, but also act as second messengers of oxidative stress, having relatively extended lifespans. Their effects might be further enhanced with aging, as their concentrations in cells and biological fluids increase with age. Since the involvement and the role of lipid peroxidation-derived aldehydes, particularly of 4-hydroxynonenal (HNE), in neurodegenerations, inflammation, and cancer, has been discussed in several excellent recent reviews, in the present one we focus on the involvement of reactive aldehydes in other age-related disorders: osteopenia, sarcopenia, immunosenescence and myelodysplastic syndromes. In these aging-related disorders, characterized by increases of oxidative stress, both HNE and malondialdehyde (MDA) play important pathogenic roles. These aldehydes, and HNE in particular, can form adducts with circulating or cellular proteins of critical functional importance, such as the proteins involved in apoptosis in muscle cells, thus leading to their functional decay and acceleration of their molecular turnover and functionality. We suggest that a major fraction of the toxic effects observed in age-related disorders could depend on the formation of aldehyde-protein adducts. New redox proteomic approaches, pinpointing the modifications of distinct cell proteins by the aldehydes generated in the course of oxidative stress, should be extended to these age-associated disorders, to pave the way to targeted therapeutic strategies, aiming to alleviate the burden of morbidity and mortality associated with these disturbances.

Deficiency of aldose reductase exacerbates early pressure overload-induced cardiac dysfunction and autophagy in mice

Pathological cardiac hypertrophy is associated with the accumulation of lipid peroxidation-derived aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) and acrolein in the heart. These aldehydes are metabolized via several pathways, of which aldose reductase (AR) represents a broad-specificity route for their elimination. We tested the hypothesis that by preventing aldehyde removal, AR deficiency accentuates the pathological effects of transverse aortic constriction (TAC). We found that the levels of AR in the heart were increased in mice subjected to TAC for 2 weeks. In comparison with wild-type (WT), AR-null mice showed lower ejection fraction, which was exacerbated 2 weeks after TAC. Levels of atrial natriuretic peptide and myosin heavy chain were higher in AR-null than in WT TAC hearts. Deficiency of AR decreased urinary levels of the acrolein metabolite, 3-hydroxypropylmercapturic acid. Deletion of AR did not affect the levels of the other aldehyde-metabolizing enzyme - aldehyde dehydrogenase 2 in the heart, or its urinary product - (N-Acetyl-S-(2-carboxyethyl)-l-cystiene). AR-null hearts subjected to TAC showed increased accumulation of HNE- and acrolein-modified proteins, as well as increased AMPK phosphorylation and autophagy. Superfusion with HNE led to a greater increase in p62, LC3II formation, and GFP-LC3-II punctae formation in AR-null than WT cardiac myocytes. Pharmacological inactivation of JNK decreased HNE-induced autophagy in AR-null cardiac myocytes. Collectively, these results suggest that during hypertrophy the accumulation of lipid peroxidation derived aldehydes promotes pathological remodeling via excessive autophagy, and that metabolic detoxification of these aldehydes by AR may be essential for maintaining cardiac function during early stages of pressure overload.

Continuous Infusion of Phenelzine, Cyclosporine A, or Their Combination: Evaluation of Mitochondrial Bioenergetics, Oxidative Damage, and Cytoskeletal Degradation following Severe Controlled Cortical Impact Traumatic Brain Injury in Rats.

To date, all monotherapy clinical traumatic brain injury (TBI) trials have failed, and there are currently no Food and Drug Administration (FDA)-approved pharmacotherapies for the acute treatment of severe TBI. Due to the complex secondary injury cascade following injury, there is a need to develop multi-mechanistic combinational neuroprotective approaches for the treatment of acute TBI. As central mediators of the TBI secondary injury cascade, both mitochondria and lipid peroxidation-derived aldehydes make promising therapeutic targets. Cyclosporine A (CsA), an FDA-approved immunosuppressant capable of inhibiting the mitochondrial permeability transition pore, and phenelzine (PZ), an FDA-approved monoamine oxidase inhibitor capable of scavenging neurotoxic lipid peroxidation-derived aldehydes, have both been shown to be partially neuroprotective following experimental TBI. Therefore, it follows that the combination of PZ and CsA may enhance neuroprotection over either agent alone through the combining of distinct but complementary mechanisms of action. Additionally, as the first 72 h represents a critical time period following injury, it follows that continuous drug infusion over the first 72 h following injury may also lead to optimal neuroprotective effects. This is the first study to examine the effects of a 72 h subcutaneous continuous infusion of PZ, CsA, and the combination of these two agents on mitochondrial respiration, mitochondrial bound 4-hydroxynonenal (4-HNE), and acrolein, and α-spectrin degradation 72 h following a severe controlled cortical impact injury in rats. Our results indicate that individually, both CsA and PZ are able to attenuate mitochondrial 4-HNE and acrolein, PZ is able to maintain mitochondrial respiratory control ratio and cytoskeletal integrity but together, PZ and CsA are unable to maintain neuroprotective effects.

Abundance of DNA adducts of 4-oxo-2-alkenals, lipid peroxidation-derived highly reactive genotoxins.

Reactive oxygen species and their reaction products can damage DNA to form mutagenic lesions. Among the reactive species, lipid peroxidation-derived aldehydes react with nucleobases and form bulky exocyclic adducts. Many types of aldehyde-derived DNA adducts have been characterized, identified and detected in vitro and in vivo, whereas relative quantitative and pathophysiological contributions of each adduct still remain unclear. In recent years, an abundant class of DNA adducts derived from 4-oxo-2-alkenals have been identified, in addition to classic aldehyde-derived adducts. The presence of 4-oxo-2-alkenal-derived DNA adducts associated with age-related diseases has been revealed in rodents and humans. In vitro studies have demonstrated that 4-oxo-2-alkenals, as compared with other classes of lipid peroxidation-derived aldehydes, are highly reactive with nucleobases. It has been generally recognized that 4-oxo-2-alkenals are generated through oxidative degradation of the corresponding 4-hydroperoxy-2-alkenals, homolytic degradation products of polyunsaturated fatty acid hydroperoxides. Our recent results have also shown an alternative pathway for the formation of 4-oxo-2-alkenals, in which 2-alkenals could undergo the metal-catalyzed autoxidation resulting in the formation of the corresponding 4-oxo-2-alkenals. This review summarizes the basis of the formation of lipid peroxidation-derived genotoxic aldehydes and their covalent adduction to nucleobases, especially focusing on the abundance of 4-oxo-2-alkenal-derived DNA adducts.

Stable isotope labeling by fatty acids in cell culture (SILFAC) coupled with isotope pattern dependent mass spectrometry for global screening of lipid hydroperoxide-mediated protein modifications

Lipid hydroperoxide-mediated modifications of proteins are receiving increasing attention because of their possible involvement in various degenerative diseases. These biological effects are attributed to the ability of lipid peroxidation-derived aldehydes to react with the nucleophilic sites of proteins. Here we describe a methodology involving metabolic labeling coupled with mass spectrometry-based proteomic analysis that enables global screening of lipid hydroperoxide-mediated protein modifications in a cell system. The lipidome of MCF-7 cells was labeled by incubating the cells with 1.4μM [13C18]-linoleic acid (LA) until the LA to [13C18]-LA ratio became 1:1. This approach was termed SILFAC (stable isotope labeling by fatty acids in cell culture). Analysis of the cellular phospholipids indicated that [13C18]-LA was incorporated quantitatively. The labeled cells were subjected to oxidative stress using a calcium ionophore and l-ascorbic acid, which promote the generation of reactive aldehydes from cellular LA and [13C18]-LA. After protein extraction and digestion with trypsin, isotope pattern dependent MS was used to analyze peptides modified by 1:1 ratios of the 12C and 13C aldehyde isomers. Using the current methodology, we identified the major lipid hydroperoxide-mediated modifications to proteins in MCF-7 cells without the need for chemical labeling or further affinity purification. SignificanceLipid peroxidation-derived aldehydes (LPDAs) such as 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal can readily react with proteins and peptides to produce a variety of covalent modifications and cross-linkages, resulting in protein dysfunction and altered gene regulation. Various analytical approaches have therefore been developed to detect and characterize protein modifications mediated by LPDAs. However, most of the methods are not specific for LPDA modifications or designed for proteins modified by a target aldehyde. Here we describe the coupling of stable isotope labeling by fatty acids in cell culture (SILFAC) with an isotope pattern dependent MS-based proteomic strategy to provide a global screening tool for the identification of lipid hydroperoxide-mediated protein modifications.

The Changes of Physical Parameters of Lipid Membrane caused by Lipid Peroxidation-Derived Aldehydes

Reactive oxygen species (ROS) are very important biological signaling molecules that play a pivotal role in cellular processes. Imbalance in ROS production and antioxidant protection results in oxidative stress, under which crucial biomolecules (DNA, proteins, lipids) can get damaged by ROS and their derivatives. Reactive aldehydes (RAs) are among various products of ROS interaction with unsaturated lipids. RAs are implicated in disfunctioning of various proteins. The molecular mechanisms underlying these interactions are still poorly understood. Recently a novel lipid-mediated mechanism was proposed, according to which biologically important RAs (4-hydroxy-2-nonenal, 4-hydroxy-2-hexenal and 4-oxo-2-nonenal) modified the activity of several membrane transporters. These RAs were shown to covalently modify the membrane lipid phosphatidylethanolamine (PE), causing formation of different membrane-active adducts.Here we study the changes of physical properties of lipid membranes induced by interaction of aldehydes with the lipid bilayer containing a large amount of PE molecules. We used lipid nanotubes pulled from the flat lipid bilayer to measure membrane bending rigidity and spontaneous curvature of membrane compounds. We showed that RAs caused significant reduction of the membrane bending rigidity. The observed decrement of the elasticity was associated with pronounced changes of the lipid molecular geometry and with the changes of packing density of lipids in the bilayer. The magnitude of the effect of different RAs on the transporter activity correlated with their effect on the lipid bilayer properties.