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Individuals with chronic kidney disease have elevated levels of oxidative stress and are at a significantly higher risk of skeletal fracture. Advanced glycation end products (AGEs), which accumulate in bone and compromise mechanical properties, are known to be driven in part by oxidative stress. The goal of this study was to study effects of N-acetylcysteine (NAC) on reducing oxidative stress and improving various bone parameters, most specifically mechanical properties, in an animal model of progressive CKD. Male Cy/+ (CKD) rats and unaffected littermates were untreated (controls) or treated with NAC (80 mg/kg, IP) from 30 to 35 weeks of age. Endpoint measures included serum biochemistries, assessments of systemic oxidative stress, bone morphology, and mechanical properties, and AGE levels in the bone. CKD rats had the expected phenotype that included low kidney function, elevated parathyroid hormone, higher cortical porosity, and compromised mechanical properties. NAC treatment had mixed effects on oxidative stress markers, significantly reducing TBARS (a measure of lipid peroxidation) while not affecting 8-OHdG (a marker of DNA oxidation) levels. AGE levels in the bone were elevated in CKD animals and were reduced with NAC although this did not translate to a benefit in bone mechanical properties. In conclusion, NAC failed to significantly improve bone architecture/geometry/mechanical properties in our rat model of progressive CKD.
Products of lipid peroxidation include a number of reactive lipid aldehydes such as malondialdehyde, 4-hydroxy-nonenal, 4-oxo-nonenal, and isolevuglandins (IsoLGs). Although these all contribute to disease processes, the most reactive are the IsoLGs, which rapidly adduct to lysine and other cellular primary amines, leading to changes in protein function, cross-linking and immunogenicity. Their rapid reactivity means that only IsoLG adducts, and not the unreacted aldehyde, can be readily measured. This high reactivity also makes it challenging for standard cellular defense mechanisms such as aldehyde reductases and oxidases to dispose of them before they react with proteins and other cellular amines. This led us to seek small molecule primary amines that might trap and inactivate IsoLGs before they could modify cellular proteins or other endogenous cellular amines such as phosphatidylethanolamines to cause disease. Our studies identified 2-aminomethylphenols including 2-hydroxybenzylamine as IsoLG scavengers. Subsequent studies showed that they also trap other lipid dicarbonyls that react with primary amines such as 4-oxo-nonenal and malondialdehyde, but not hydroxyalkenals like 4-hydroxy-nonenal that preferentially react with soft nucleophiles. This review describes the use of these 2-aminomethylphenols as dicarbonyl scavengers to assess the contribution of IsoLGs and other amine-reactive lipid dicarbonyls to disease and as therapeutic agents.
Copyright © 2019. Published by Elsevier Inc.
infects every niche of the human host. In response to microbial infection, vertebrates have an arsenal of antimicrobial compounds that inhibit bacterial growth or kill bacterial cells. One class of antimicrobial compounds consists of polyunsaturated fatty acids, which are highly abundant in eukaryotes and encountered by at the host-pathogen interface. Arachidonic acid (AA) is one of the most abundant polyunsaturated fatty acids in vertebrates and is released in large amounts during the oxidative burst. Most of the released AA is converted to bioactive signaling molecules, but, independently of its role in inflammatory signaling, AA is toxic to Here, we report that AA kills through a lipid peroxidation mechanism whereby AA is oxidized to reactive electrophiles that modify macromolecules, eliciting toxicity. This process is rescued by cotreatment with antioxidants as well as in a strain genetically inactivated for (USA300 mutant) that produces lower levels of reactive oxygen species. However, resistance to AA stress in the USA300 mutant comes at a cost, making the mutant more susceptible to β-lactam antibiotics and attenuated for pathogenesis in a murine infection model compared to the parental methicillin-resistant (MRSA) strain, indicating that resistance to AA toxicity increases susceptibility to other stressors encountered during infection. This report defines the mechanism by which AA is toxic to and identifies lipid peroxidation as a pathway that can be modulated for the development of future therapeutics to treat infections. Despite the ability of the human immune system to generate a plethora of molecules to control infections, is among the pathogens with the greatest impact on human health. One class of host molecules toxic to consists of polyunsaturated fatty acids. Here, we investigated the antibacterial properties of arachidonic acid, one of the most abundant polyunsaturated fatty acids in humans, and discovered that the mechanism of toxicity against proceeds through lipid peroxidation. A better understanding of the molecular mechanisms by which the immune system kills , and by which avoids host killing, will enable the optimal design of therapeutics that complement the ability of the vertebrate immune response to eliminate infections.
Copyright © 2019 Beavers et al.
Isolevuglandins are 4-ketoaldehydes formed by peroxidation of arachidonic acid. Isolevuglandins react rapidly with primary amines including the lysyl residues of proteins to form irreversible covalent modifications. This review highlights evidence for the potential role of isolevuglandin modification in the disease processes, especially atherosclerosis, and some of the tools including small molecule dicarbonyl scavengers utilized to assess their contributions to disease.
Copyright © 2018. Published by Elsevier Inc.
Reactive oxygen species (ROS) are formed in mitochondria during electron transport and energy generation. Elevated levels of ROS lead to increased amounts of mitochondrial DNA (mtDNA) damage. We report that levels of M1dG, a major endogenous peroxidation-derived DNA adduct, are 50-100-fold higher in mtDNA than in nuclear DNA in several different human cell lines. Treatment of cells with agents that either increase or decrease mitochondrial superoxide levels leads to increased or decreased levels of M1dG in mtDNA, respectively. Sequence analysis of adducted mtDNA suggests that M1dG residues are randomly distributed throughout the mitochondrial genome. Basal levels of M1dG in mtDNA from pulmonary microvascular endothelial cells (PMVECs) from transgenic bone morphogenetic protein receptor 2 mutant mice (BMPR2R899X) (four adducts per 106 dG) are twice as high as adduct levels in wild-type cells. A similar increase was observed in mtDNA from heterozygous null (BMPR2+/-) compared to wild-type PMVECs. Pulmonary arterial hypertension is observed in the presence of BMPR2 signaling disruptions, which are also associated with mitochondrial dysfunction and oxidant injury to endothelial tissue. Persistence of M1dG adducts in mtDNA could have implications for mutagenesis and mitochondrial gene expression, thereby contributing to the role of mitochondrial dysfunction in diseases.
Parkinson's disease (PD) is the result of complex interactions between genetic and environmental factors. Two chemically distinct environmental stressors relevant to PD are the metal manganese and the pesticide rotenone. Both are thought to exert neurotoxicity at least in part via oxidative stress resulting from impaired mitochondrial activity. Identifying shared mechanism of action may reveal clues towards an understanding of the mechanisms underlying PD pathogenesis. Here we compare the effects of manganese and rotenone in human-induced pluripotent stem cells-derived postmitotic mesencephalic dopamine neurons by assessing several different oxidative stress endpoints. Manganese, but not rotenone caused a concentration and time-dependent increase in intracellular reactive oxygen/nitrogen species measured by quantifying the fluorescence of oxidized chloromethyl 2',7'-dichlorodihydrofluorescein diacetate (DCF) assay. In contrast, rotenone but not manganese caused an increase in cellular isoprostane levels, an indicator of lipid peroxidation. Manganese and rotenone both caused an initial decrease in cellular reduced glutathione; however, glutathione levels remained low in neurons treated with rotenone for 24 h but recovered in manganese-exposed cells. Neurite length, a sensitive indicator of overall neuronal health was adversely affected by rotenone, but not manganese. Thus, our observations suggest that the cellular oxidative stress evoked by these 2 agents is distinct yielding unique oxidative stress signatures across outcome measures. The protective effect of rasagiline, a compound used in the clinic for PD, had negligible impact on any of oxidative stress outcome measures except a subtle significant decrease in manganese-dependent production of reactive oxygen/nitrogen species detected by the DCF assay.
© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: email@example.com.
Determining the impact of lipid electrophile-mediated protein damage that occurs during oxidative stress requires a comprehensive analysis of electrophile targets adducted under pathophysiological conditions. Incorporation of ω-alkynyl linoleic acid into the phospholipids of macrophages prior to activation by Kdo-lipid A, followed by protein extraction, click chemistry, and streptavidin affinity capture, enabled a systems-level survey of proteins adducted by lipid electrophiles generated endogenously during the inflammatory response. Results revealed a dramatic enrichment for membrane and mitochondrial proteins as targets for adduction. A marked decrease in adduction in the presence of MitoTEMPO demonstrated a primary role for mitochondrial superoxide in electrophile generation and indicated an important role for mitochondria as both a source and target of lipid electrophiles, a finding that has not been revealed by prior studies using exogenously provided electrophiles.
The cellular production of free radicals or reactive oxygen species (ROS) can lead to protein, lipid or DNA modifications and tumor formation. The cellular lipids undergo structural changes through the actions of enzymes (e.g. cyclooxygenases) or free radicals to form a class of compounds called Isolevuglandins (IsoLGs). The recruitment and continued exposure of tissue to ROS and IsoLGs causes increased cell proliferation, mutagenesis, loss of normal cell function and angiogenesis. The elevated concentration of ROS in cancerous tissues suggests that these mediators play an important role in cancer development. We hypothesized that tumors with elevated ROS levels would similarly possess an increased concentration of IsoLGs when compared with normal tissue. Using D11, an ScFv recombinant antibody specific for IsoLGs, we utilized immunohistochemistry to visualize the presence of IsoLG in human tumors compared to normal adjacent tissue (NAT) to the same tumor. We found that IsoLG concentrations were elevated in human breast, colon, kidney, liver, lung, pancreatic and tongue tumor cells when compared to NAT and believe that IsoLGs can be used as a gauge indicative of lipid peroxidation in tumors.
Copyright © 2017 Elsevier Inc. All rights reserved.
AIMS - Products of lipid oxidation, such as 4-hydroxynonenal (4-HNE), are key activators of hepatic stellate cells (HSC) to a pro-fibrogenic phenotype. Isolevuglandins (IsoLG) are a family of acyclic γ-ketoaldehydes formed through oxidation of arachidonic acid or as by-products of the cyclooxygenase pathway. IsoLGs are highly reactive aldehydes which are efficient at forming protein adducts and cross-links at concentrations 100-fold lower than 4-hydroxynonenal. Since the contribution of IsoLGs to liver injury has not been studied, we synthesized 15-E-IsoLG and used it to investigate whether IsoLG could induce activation of HSC.
RESULTS - Primary human HSC were exposed to 15-E-IsoLG for up to 48h. Exposure to 5μM 15-E-IsoLG in HSCs promoted cytotoxicity and apoptosis. At non-cytotoxic doses (50 pM-500nM) 15-E-IsoLG promoted HSC activation, indicated by increased expression of α-SMA, sustained activation of ERK and JNK signaling pathways, and increased mRNA and/or protein expression of cytokines and chemokines, which was blocked by inhibitors of JNK and NF-kB. In addition, IsoLG promoted formation of reactive oxygen species, and induced an early activation of ER stress, followed by autophagy. Inhibition of autophagy partially reduced the pro-inflammatory effects of IsoLG, suggesting that it might serve as a cytoprotective response.
INNOVATION - This study is the first to describe the biological effects of IsoLG in primary HSC, the main drivers of hepatic fibrosis.
CONCLUSIONS - IsoLGs represent a newly identified class of activators of HSC in vitro, which are biologically active at concentrations as low as 500 pM, and are particularly effective at promoting a pro-inflammatory response and autophagy.
Copyright © 2016. Published by Elsevier Inc.
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.