Levels Of Protein Adducts Research Articles

The Anti-Inflammatory Action of Cannabigerol Accompanied by the Antioxidant Effect of 3-O-ethyl Ascorbic Acid in UVA-Irradiated Human Keratinocytes.

Excessive daily exposure of human skin to natural UVA radiation leads to impaired redox homeostasis in epidermal keratinocytes, resulting in changes in their proteome. Commonly used antioxidants usually exhibit protection in a narrowed range, which makes it necessary to combine their effects. Therefore, the aim of this study was to analyze the protective effect of cannabigerol (CBG) and 3-O-ethyl ascorbic acid (EAA), used separately and together, on the proteomic profile of UVA irradiated keratinocytes. Proteomic analysis with the use of the Q Exactive HF mass spectrometer, combined with biostatistic tests, performed on UVA-irradiated keratinocytes indicated enhanced and lowered expression of 186 and 160 proteins, respectively. CBG treatment after UVA irradiation reduced these numbers to 110 upregulated and 49 downregulated proteins, while EAA eliminated all these changes. CBG completely eliminated the UV-induced effect on the expression of pro-inflammatory proteins and significantly increased the level of proteins responsible for cellular locomotion. On the other hand, CBG reduced the level of UVA-induced 4-hydroxynonenal protein adducts fivefold, whereas EAA had no effect on this modification. At the same time, CBG and EAA did not modify the expression/structure of proteins in relation to the nonirradiated control keratinocytes in the case of an unaccompanied use or slightly modified the protein profile when used in a mixture. The combined protective effects of CBG on protein structure and EAA on protein expression profile allowed us to obtain a wider protection of cells against UVA radiation, compared with when the compounds were used alone. SIGNIFICANCE STATEMENT: Proteomic analysis of human skin cells allows to conclude that 3-O-ethyl ascorbic acid eliminates UVA-induced changes in the expression of keratinocyte proteins, while cannabigerol significantly reduces 4-hydroxynonenal protein adducts. The combined protective effects of cannabigerol on protein structure and of 3-O-ethyl ascorbic acid on protein expression profile allowed to obtain a wider protection of cells against UVA radiation.

Identification of intestinal metabolic activation of loganin generated dialdehyde reactive intermediates improves intestinal bile salt hydrolase activities

Loganin is an iridoid with potent pharmacological effects. Loganin contains a hemiacetal structure and can convert to dialdehyde intermediates after deglycosylation. We hypothesized that the metabolites of loganin with hemiacetal can generate reactive dialdehyde intermediates. This study aims to characterize the metabolic profiling of loganin and especially for the unstable dialdehyde intermediates by using ultra-performance liquid chromatograph-quadrupole orbitrap mass spectrometry. In this study, a total of 26 stable metabolites were identified in loganin-treated rats. Loganin underwent different metabolism in the intestine and liver, which was confirmed mainly by the metabolites in the hepatic portal vein. In the intestine, the major metabolic pathways were ester hydrolysis and deglycosylation, followed by methylation and dehydrogenation. The hepatic metabolism pathways were hydrogenation, hydroxylation, glucuronidation, and sulfonation. The circulating metabolites with high abundance were mainly derived from intestinal metabolism. Importantly, 11 unstable dialdehyde intermediates of loganin were identified and described for the first time. The dialdehyde intermediates were identified by their dihydropyridine conjugates with amino acids. The dialdehyde intermediates were mainly produced in the intestine. The dialdehyde intermediates enable covalent modification of intestinal proteins. Loganin can up-regulate the activity of intestinal bile salt hydrolase (BSH), catalyzing bile acid metabolism. The level of protein adducts was positively associated with BSH activity, indicating dialdehyde intermediates played a key role in the up-regulation of BSH activities. In conclusion, this study not only demonstrates the characteristic metabolic fate of loganin but also facilitates the understanding of the pharmacologic effects of dialdehyde intermediates.

Protective effect of genistein pre-treatment on paraquat hepatotoxicity in rats

Paraquat (PQ), an herbicide widely used in agriculture, is considered a highly toxic compound. In hepatocytes, P-glycoprotein (P-gp/Abcb1) is a canalicular transporter involved in PQ extrusion from the cell. Previously, we demonstrated that genistein (GNT) induces P-gp in rat liver. In this study, the protective role of GNT pretreatment towards hepatic damage in a model of acute intoxication with PQ in rats, was investigated. Wistar rats were randomized in 4 groups: Control, GNT (5 mg/kg/day sc, 4 days), PQ (50 mg/kg/day ip, last day) and GNT+ PQ. Hepatic lipoperoxidation (LPO) was evaluated by the thiobarbituric acid reactive substances method. Hepatic levels of 4-hydroxynonenal protein adducts (4-HNEp-add) and glutathione-S-transferase alpha (GSTα) protein expression were evaluated by Western blotting. Hepatic glutathione levels and plasma levels of alanine transaminase (ALT) and aspartate transaminase (AST) were also measured. Biliary excretion of PQ was studied in vivo and in isolated perfused liver. PQ was quantified by HPLC. PQ significantly increased AST and ALT activities, malondialdehyde and 4-HNEp-add levels, whereby pretreatment with GNT ameliorated this effect. PQ biliary excretion remained unchanged after treatments in both experimental models. Hepatic GSTα expression was augmented in GNT group. GNT pretreatment increased hepatic glutathione levels in PQ + GNT group. These results agree with the lower content of 4-HNEp-adds in GNT + PQ group respect to PQ group. Unexpectedly, increased activity of P-gp did not enhance PQ biliary excretion. Thus, GNT protective mechanism is likely through the induction of GSTα which results in increased 4-HNE metabolism before formation of protein adducts.

Novel Spray Dried Algae-Rosemary Particles Attenuate Pollution-Induced Skin Damage.

The present study investigated the effect of spray-dried algae-rosemary particles against pollution-induced damage using ex-vivo human biopsies exposed to diesel engine exhaust (DEE). For this, the complexation of hydroalcoholic rosemary extract with Chlorella (RCH) and Spirulina (RSP) protein powders was conducted. The process efficiency and concentration of rosmarinic acid (RA), carnosic acid (CA), and carnosol (CR) phenolic compounds of both products were compared. The RSP spray-dried production was more efficient, and RSP particles presented higher CR and CA and similar RA concentrations. Therefore, spray-dried RSP particles were prioritized for the preparation of a gel formulation that was investigated for its ability to mitigate pollution-induced skin oxinflammatory responses. Taken altogether, our ex-vivo data clearly demonstrated the ability of RSP gel to prevent an oxinflammatory phenomenon in cutaneous tissue by decreasing the levels of 4-hydroxynonenal protein adducts (4HNE-PA) and active matrix metalloproteinase-9 (MMP-9) as well as by limiting the loss of filaggrin induced by DEE exposure. Our results suggest that the topical application of spirulina-rosemary gel is a good approach to prevent pollution-induced skin aging/damage.

Dynamic and Temporal Transcriptomic Analysis Reveals Ferroptosis-Mediated Antileukemia Activity of S-Dimethylarsino-Glutathione: Insights into Novel Therapeutic Strategy

Dynamic and Temporal Transcriptomic Analysis Reveals Ferroptosis-Mediated Antileukemia Activity of S-Dimethylarsino-Glutathione: Insights into Novel Therapeutic Strategy

Quantitation of Nitration, Chlorination, and Oxidation in Hemoglobin of Breast Cancer Patients by Nanoflow Liquid Chromatography Tandem Mass Spectrometry.

Cells are continually exposed to endogenous reactive oxygen, nitrogen, and halogen species, causing damage to biomolecules. Among them, peroxynitrite and hypochlorous acid are not only oxidants but also biological nitrating and chlorinating agents, leading to the formation of 3-nitrotyrosine and 3-chlorotyrosine, respectively, in proteins. 3-Nitrotyrosine has been detected in vivo under several pathophysiological conditions, including breast cancer. Studies show that the concentrations of 3-nitrotyrosine in plasma proteins and platelets were significantly elevated in breast cancer patients. Compared to blood serum albumin, hemoglobin adducts represent biomonitoring of exposure with a longer lifetime. In this study, human hemoglobin was freshly isolated from blood and digested into peptides with trypsin, and the levels of protein adducts, including nitration, nitrosylation, and chlorination of tyrosine as well as oxidation of methionine residues, were simultaneously quantified by nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) with selected reaction monitoring. The results demonstrated that the relative extents of nitration at α-Tyr-42 and β-Tyr-130, nitrosylation at α-Tyr-24, and chlorination at α-Tyr-24 and β-Tyr-130 are significantly higher in globin of 25 breast cancer patients compared to those in 25 healthy subjects (p < 0.05). In particular, nitration at α-Tyr-42 and chlorination at α-Tyr-24 showed the area under the receiver operating characteristic curve of >0.8. While the age of the subjects is correlated with the extents of some of these adducts, the body mass index does not have an effect on any of them. Starting with 1 drop of blood, our results indicated that this highly sensitive and specific nanoLC-NSI/MS/MS is useful in investigating the role of reactive nitrogen oxide species and reactive chlorine species in the etiology of breast cancer.

Tempol improves redox status in mdx dystrophic diaphragm muscle.

Oxidative stress is a critical element in relationship to the pathophysiology of Duchenne muscular dystrophy (DMD). In the mice the diaphragm (DIA) is most resembles the dystrophic human pathology. In this study we have evaluated the consequences of a synthetic antioxidant (tempol) on oxidative stress parameters in the DIA muscle of mdx mice. The mdx mice were separated into two groups: mdx, the control group receiving intraperitoneal (i.p.) injections of saline solution (100µL), and mdxT, the treated group receiving i.p. injections of tempol (100mg/kg). The tempol-treated group showed reduced oxidative stress markers, decreasing the dihydroethidium reaction (DHE) area; autofluorescent lipofuscin granules; and 4-hydroxynonenal (4-HNE)-protein adduct levels. DIA muscle of mdx mice. At the same time, the manganese-superoxide dismutase 2 (SOD2) levels were increased in the tempol-treated group. In addition, the tempol-treated group showed reduced levels of glutathione-disulphide reductase (GSR), glutathione peroxidase 1 (GPx1) and catalase (CAT) in immunoblots. The tempol-treated group has also shown lower relative gene expression of SOD1, CAT and GPx than the non-treated group. Our data demonstrated that tempol treatment reduced oxidant parameters and increased anti-oxidant SOD2 levels in the DIA muscle of mdx mice, which may contribute to the normalization of the redox homeostasis of dystrophic muscles.

ALKBH7 mediates necrosis via rewiring of glyoxal metabolism.

Alkb homolog 7 (ALKBH7) is a mitochondrial α-ketoglutarate dioxygenase required for DNA alkylation-induced necrosis, but its function and substrates remain unclear. Herein, we show ALKBH7 regulates dialdehyde metabolism, which impacts the cardiac response to ischemia-reperfusion (IR) injury. Using a multi-omics approach, we find no evidence ALKBH7 functions as a prolyl-hydroxylase, but we do find Alkbh7-/- mice have elevated glyoxalase I (GLO-1), a dialdehyde detoxifying enzyme. Metabolic pathways related to the glycolytic by-product methylglyoxal (MGO) are rewired in Alkbh7-/- mice, along with elevated levels of MGO protein adducts. Despite greater glycative stress, hearts from Alkbh7-/- mice are protected against IR injury, in a manner blocked by GLO-1 inhibition. Integrating these observations, we propose ALKBH7 regulates glyoxal metabolism, and that protection against necrosis and cardiac IR injury bought on by ALKBH7 deficiency originates from the signaling response to elevated MGO stress.

CKD Increases Carbonylation of HDL and Is Associated with Impaired Antiaggregant Properties.

CKD is associated with increased oxidative stress that correlates with occurrence of cardiovascular events. Modifications induced by increased oxidative stress particularly affect circulating lipoproteins such as HDL that exhibit antiatheromatous and antithrombotic properties in vitro. To explore the specific role of oxidative modifications of HDL in CKD and their effect on the platelet-targeting antiaggregant properties of HDL, we used a CKD (5/6 nephrectomy) rabbit model. For ex vivo assessment of the antiaggregant properties of HDL, we collected blood samples from 15 healthy volunteers, 25 patients on hemodialysis, and 20 on peritoneal dialysis. We analyzed malondialdehyde, 4-hydroxynonenal (HNE), and 4-hydroxy-2-hexenal protein adduct levels. Platelet aggregation and activation were assessed by aggregometry, thromboxane B2 assay, or FACS. We modified HDL from controls by incubating it overnight at 37°C with 100 µM of HNE. HDL from CKD rabbits and patients on hemodialysis had HNE adducts. The percentage of platelet aggregation or activation induced by collagen was significantly higher when platelets were incubated with HDL from CKD rabbit and hemodialysis groups than with HDL from the control group. In both rabbits and humans, platelet aggregation and activation were significantly higher in the presence of HNE-modified HDL than with HDL from their respective controls. Incubation of platelets with a blocking antibody directed against CD36 or with a pharmacologic inhibitor of SRC kinases restored the antiaggregative phenotype in the presence of HDL from CKD rabbits, patients on hemodialysis and peritoneal dialysis, and HNE-modified HDL. HDL from CKD rabbits and patients on hemodialysis exhibited an impaired ability to inhibit platelet aggregation, suggesting that altered HDL properties may contribute to the increased cardiovascular risk in this population.

Protective effects of a comprehensive topical antioxidant against ozone-induced damage in a reconstructed human skin model

Tropospheric ozone (O3) is a source of oxidative stress. This study examined the ability of a topical antioxidant (WEL-DS) to inhibit O3-mediated damage in a human epidermal skin model. Four groups of tissues (N = 24) were compared: Group 1 (control) were untreated and unexposed; Group 2 were untreated and exposed to O3 (0.4 ppm, 4 h); Group 3 were pretreated with WEL-DS and unexposed; Group 4 were pretreated with WEL-DS and exposed to O3 (0.4 ppm, 4 h). Pretreated tissues were topically treated with 20 uL of WEL-DS and incubated for up to 20 h at 37 °C [humidified, 5% carbon dioxide (CO2)]. After 24 h, tissues were re-treated with WEL-DS and exposed to O3. Tissues were evaluated for Reactive Oxygen Species (ROS), hydrogen peroxide (H2O2), 4-hydroxynonenal (4-HNE) protein adducts, NF-κB p65 response and histology. In O3-exposed groups, WEL-DS significantly inhibited ROS formation vs. untreated tissues (p < 0.05). Pretreatment with WEL-DS inhibited H2O2 production vs. untreated tissues (p < 0.05), and decreased NF-κB p65 transcription factor signal. Oxidative stress induction in O3-exposed tissues was confirmed by increased levels of 4-HNE protein adducts (marker of lipid peroxidation); WEL-DS application reduced this effect. WEL-DS inhibited damage in tissues exposed to O3 with no significant changes in epidermal structure. A comprehensive topical antioxidant significantly diminished O3-induced oxidative damage in a human epidermal skin model.

Alterations of mitochondrial bioenergetics, dynamics, and morphology support the theory of oxidative damage involvement in autism spectrum disorder.

Autism spectrum disorder (ASD) has been hypothesized to be a result of the interplay between genetic predisposition and increased vulnerability to early environmental insults. Mitochondrial dysfunctions appear also involved in ASD pathophysiology, but the mechanisms by which such alterations develop are not completely understood. Here, we analyzed ASD primary fibroblasts by measuring mitochondrial bioenergetics, ultrastructural and dynamic parameters to investigate the hypothesis that defects in these pathways could be interconnected phenomena responsible or consequence for the redox imbalance observed in ASD. High levels of 4-hydroxynonenal protein adducts together with increased NADPH (nicotinamide adenine dinucleotide phosphateoxidase) activity and mitochondrial superoxide production coupled with a compromised antioxidant response guided by a defective Nuclear Factor Erythroid 2-Related Factor 2 pathway confirmed an unbalanced redox homeostasis in ASD. Moreover, ASD fibroblasts showed overactive mitochondrial bioenergetics associated with atypical morphology and altered expression of mitochondrial electron transport chain complexes and dynamics-regulating factors. We suggest that many of the changes observed in mitochondria could represent compensatory mechanisms by which ASD cells try to adapt to altered energy demand, possibly resulting from a chronic oxinflammatory status.

Astaxanthin Ameliorated Parvalbumin-Positive Neuron Deficits and Alzheimer's Disease-Related Pathological Progression in the Hippocampus of AppNL-G-F/NL-G-F Mice.

Growing evidence suggests that oxidative stress due to amyloid β (Aβ) accumulation is involved in Alzheimer’s disease (AD) through the formation of amyloid plaque, which leads to hyperphosphorylation of tau, microglial activation, and cognitive deficits. The dysfunction or phenotypic loss of parvalbumin (PV)-positive neurons has been implicated in cognitive deficits. Astaxanthin is one of carotenoids and known as a highly potent antioxidant. We hypothesized that astaxanthin’s antioxidant effects may prevent the onset of cognitive deficits in AD by preventing AD pathological processes associated with oxidative stress. In the present study, we investigated the effects of astaxanthin intake on the cognitive and pathological progression of AD in a mouse model of AD. The AppNL-G-F/NL-G-F mice were fed with or without astaxanthin from 5-to-6 weeks old, and cognitive functions were evaluated using a Barnes maze test at 6 months old. PV-positive neurons were investigated in the hippocampus. Aβ42 deposits, accumulation of microglia, and phosphorylated tau (pTau) were immunohistochemically analyzed in the hippocampus. The hippocampal anti-oxidant status was also investigated. The Barnes maze test indicated that astaxanthin significantly ameliorated memory deficits. Astaxanthin reduced Aβ42 deposition and pTau-positive areal fraction, while it increased PV-positive neuron density and microglial accumulation per unit fraction of Aβ42 deposition in the hippocampus. Furthermore, astaxanthin increased total glutathione (GSH) levels, although 4-hydroxy-2,3-trans-nonenal (4-HNE) protein adduct levels (oxidative stress marker) remained high in the astaxanthin supplemented mice. The results indicated that astaxanthin ameliorated memory deficits and significantly reversed AD pathological processes (Aβ42 deposition, pTau formation, GSH decrease, and PV-positive neuronal deficits). The elevated GSH levels and resultant recovery of PV-positive neuron density, as well as microglial activation, may prevent these pathological processes.

Methylglyoxal Acts as a Tumor-Promoting Factor in Anaplastic Thyroid Cancer.

Methylglyoxal (MG) is a potent inducer of advanced glycation end products (AGEs). MG, long considered a highly cytotoxic molecule with potential anticancer value, is now being re-evaluated to a protumorigenic agent in some malignancies. Anaplastic thyroid cancer (ATC) is an extremely aggressive and highly lethal cancer for which conventional therapies have proved ineffective. Successful therapeutic intervention in ATC is undermined by our poor understanding of its molecular etiology. In the attempt to understand the role of MG in ATC aggressiveness, we used immunohistochemistry to examine the level of MG protein adducts in ATC and slow-growing papillary thyroid cancer (PTC). We detected a high level of MG adducts in ATC compared to PTC ones, suggesting a protumor role for MG-mediated dicarbonyl stress in ATC. Accordingly, MG adduct accumulation in ATC cells in vitro was associated with a marked mesenchymal phenotype and increased migration/invasion, which were both reversed by aminoguanidine (AG)—a scavenger of MG—and resveratrol—an activator of Glyoxalase 1 (Glo1), the key metabolizing enzyme of MG. Our study represents the first demonstration that MG, via AGEs, acts as a tumor-promoting factor in ATC and suggests that MG scavengers and/or Glo1 activators merit investigations as potential therapeutic strategies for this malignancy.

S-metolachlor promotes oxidative stress in green microalga Parachlorella kessleri - A potential environmental and health risk for higher organisms

The estimation of the toxic influences of herbicide products on non-target aquatic organisms is essential for evaluation of environmental contamination. We assessed the effects of the herbicide S-metolachlor (S-MET) on unicellular green microalga Parachlorella kessleri during 4–72 in vitro exposure to concentrations in the range 2–200μg/L. The results have shown that S-MET had a significant effect on algae, even in doses 10 and 20 times lower than the EC50 values obtained for P. kessleri (EC50-72h=1090μg/L). It generates reactive oxygen species in algae, decreases their growth and photosynthetic pigment concentration, changes their ultrastructure and alters the cellular antioxidant defence capacities. The levels of protein adducts with the reactive aldehyde 4-hydroxy-2-nonenal (HNE), the end-product of lipid peroxidation, were significantly elevated in S-MET treated cells revealing the insufficient effectiveness of P. kessleri antioxidant mechanisms and persistent lipid peroxidation. Since algae are fundamental aquatic food component, the damaged algal cells, still capable of dividing while having persistently increased content of HNE upon S-MET contamination could represent an important environmental toxic factor that might further affect higher organisms in the food chain.

Effects of vitamin D on insulin resistance and myosteatosis in diet-induced obese mice.

Epidemiological studies pointed out to a strong association between vitamin D deficiency and type 2 diabetes prevalence. However, the role of vitamin D supplementation in the skeletal muscle, a tissue that play a crucial role in the maintenance of glucose homeostasis, has been scarcely investigated so far. On this basis, this study aimed to evaluate the effect of vitamin D supplementation in a murine model of diet-induced insulin resistance with particular attention to the effects evoked on the skeletal muscle. Male C57BL/6J mice (n = 40) were fed with a control or a High Fat-High Sugar (HFHS) diet for 4 months. Subsets of animals were treated for 2 months with vitamin D (7 μg·kg-1, i.p. three times/week). HFHS diet induced body weight increase, hyperglycemia and impaired glucose tolerance. HFHS animals showed an impaired insulin signaling and a marked fat accumulation in the skeletal muscle. Vitamin D reduced body weight and improved systemic glucose tolerance. In addition, vitamin D restored the impaired muscle insulin signaling and reverted myosteatosis evoked by the diet. These effects were associated to decreased activation of NF-κB and lower levels of TNF-alpha. Consistently, a significantly decreased activation of the SCAP/SREBP lipogenic pathway and lower levels of CML protein adducts and RAGE expression were observed in skeletal muscle of animals treated with vitamin D.Collectively, these data indicate that vitamin D-induced selective inhibition of signaling pathways (including NF-κB, SCAP/SREBP and CML/RAGE cascades) within the skeletal muscle significantly contributed to the beneficial effects of vitamin D supplementation against diet-induced metabolic derangements.

Magnolol protects pancreatic β-cells against methylglyoxal-induced cellular dysfunction

Chronic hyperglycemia aggravates insulin resistance, in part due to increased formation of advanced glycation end-products (AGEs). Methylglyoxal (MG), a major precursor of AGEs, accumulates abnormally in various tissues and organs and participates in oxidative damage. We investigated the insulinotropic benefits of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, in pancreatic β-cells exposed to MG in vitro. When exposed to cytotoxic levels of MG for 48 h, RIN-m5F β-cells exhibited a significant loss of viability and impaired insulin secretion, whereas pretreatment with magnolol protected against MG-induced cell death and decreased insulin secretion. Moreover, magnolol increased the expression of genes involved in β-cell survival and function, including Ins2 and PDX1. Furthermore, magnolol increased the levels of AMPK phosphorylation, SIRT1, and PGC1α in RIN-5F β-cells. In addition, magnolol increased the activity of glyoxalase I and decreased the levels of MG-modified protein adducts, which suggests that magnolol protects against MG-induced protein glycation. Taken together, the results indicate the potential application of magnolol as an intervention against MG-induced hyperglycemia.

Isotypes of autoantibodies against differentially expressed novel malondialdehyde-modified peptide adducts in serum of Taiwanese women with rheumatoid arthritis

This study identified and validated four differentially expressed novel malondialdehyde (MDA)-modified peptide adducts and evaluated autoantibodies against native and MDA-modified peptides among Taiwanese women patients with rheumatoid arthritis (RA), osteoarthritis (OA) and healthy controls (HCs). Ig kappa chain C region76–99, alpha-1-antitrypsin284–298, alpha-2-macroglobulin824–841, and apolipoprotein B-1004022–4040 exhibiting 2-fold differences in relative modification ratios were identified by concanavalin A (Con A) affinity chromatography, 1D SDS-PAGE, in-gel digestion, nano-LC/MS/MS and nano-LC/MS using pooled serum-derived Con A-captured proteins from 9 RA and 9 age-matched HCs. Furthermore, the levels of proteins, serum MDA, and MDA-modified protein adducts were further validated against individual serum from 20 RA and 20 HCs, and autoantibodies against native and their MDA-modified peptides used 45 RA, 30 OA and 45 HCs. Levels of serum MDA and MDA-modified protein adducts were significantly higher in RA than HCs but protein levels were not significantly different. Serum Igs G and M against MDA-modified peptides showed better diagnostic performance in differentiating among patients with RA, OA and HCs, with an area under the receiver operating characteristic curve of 0.96–0.98, sensitivity of 88.9%–97.8%, and specificity of 88.9%–100%. Autoantibodies against MDA-modified epitopes become useful clinical biomarkers for RA. Biological significanceBy using a label-free relative quantitative proteomic analysis of concanavalin A (Con A)-bound serum samples, the current study discovered and validated malondialdehyde (MDA)-modified peptide adducts as novel biomarkers for differentiating between rheumatoid arthritis (RA) patients and healthy controls (HCs). In addition, the serum levels of MDA, proteins, and MDA-modified protein adducts as well as the MDA modification of proteins were determined. Isotypes of autoantibodies against MDA-modified peptide adducts can be used as serological biomarkers for further discriminating among RA patients, osteoarthritis patients and HCs. This strategy can become the basis for identifying potential diagnostic and pathological biomarkers for RA.

The sphingosine 1-phosphate breakdown product, (2E)-hexadecenal, forms protein adducts and glutathione conjugates in vitro

Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes such as cell proliferation and apoptosis, can be irreversibly cleaved by S1P lyase, yielding phosphoethanolamine and (2E)-hexadecenal (2EHD). The latter metabolite, an α,β-unsaturated fatty aldehyde, may be susceptible to nucleophilic attack by cellular biomolecules. Hence, we studied whether 2EHD forms reaction products with GSH and proteins in vitro. Using LC-MS/MS and stable isotopically labeled reference material, we identified a total of nine novel reaction products of 2EHD in a cell-free approach: two GSH conjugates and seven l-amino acid adducts. Both GSH conjugates were also found in HepG2 cell lysates incubated with 2EHD. Likewise, we detected four out of seven amino acid adducts released from the model protein, BSA, and proteins extracted from HepG2 cells. On this occasion, the 2EHD Michael adduct with l-histidine proved to be the most prominent adduct. Most interestingly, inhibition of the enzymatically driven oxidative degradation of 2EHD resulted in increased levels of both GSH conjugates and protein adducts in HepG2 cell lysates. Hence, our data provide new insights into sphingolipid metabolism and will be useful to investigate certain disorders linked to an impaired fatty aldehyde metabolism in more detail.

Oxidative stress with tau hyperphosphorylation in memory impaired 1,2-diacetylbenzene-treated mice

Long-term exposure to organic solvent may be related to the incidence of neuronal diseases, such as, Alzheimer’s disease, depression, multiple sclerosis, dementia, Parkinson’s disease. Previously, the authors reported 1,2-diacetylbenzene (DAB; a neurotoxic metabolite of 1,2-diethylbenzene) causes central and peripheral neuropathies that lead to motor neuronal deficits. Furthermore, it is known DAB increases oxidative stress and protein adduct levels and impairs hippocampal neurogenesis in mice. The authors examined the relevance of oxidative stress and tau hyperphosphorylation in the hippocampus. Five-week-old male C57BL/6 mice were treated with 1 or 5mg/kg/day DAB for 2weeks. Neither overall body weight increases nor behavioral differences were observed after treatment, but kidney and liver weights decreased. Increased ROS production, activated glycogen synthase kinase-3β (GSK-3β) and tau hyperphosphorylation were observed in hippocampal homogenates. To assess memory impairment, the Morris Water Maze was used. Animals in the DAB-treated groups took longer to reach the platform. Movement patterns of DAB treated mice were more complicated and their swimming speeds were lower than those of controls. When SHSY5Y neuroblastoma cells were pretreated with NAC (an antioxidant) or a GSK-3β inhibitor, the expression of active GSK-3β and tau hyperphosphorylation were reduced. These results suggest ROS produced by DAB causes tau hyperphosphorylation via GSK-3β phosphorylation and it might be related to impaired memory deficit.

P 138 - A correlation between redox imbalance and altered mitochondrial quality control pathway in Autistic Spectrum Disorder

The Autistic Spectrum Disorder (ASD) refers to a range of conditions classified as neurodevelopmental disorders characteristic of the early childhood. Over the last 50 years, considerable efforts have been made to understand pathogenesis of ASD: it is generally accepted that a lot of causes could trigger the disease. Since growing evidences described an association between oxidative stress and mitochondrial dysfunction in ASD, we investigated the possible defects of mitochondrial quality control pathway as contributing factors to the altered redox homeostasis. Using freshly skin fibroblasts isolated from ASD patients and healthy subjects, we demonstrated in autistic cells an increased levels of 4HNE protein adducts - a major secondary product of lipid peroxidation - and catalase, one of the main enzymes of the cellular defensive system. Transmission electron microscope (TEM) analysis demonstrated an altered mitochondrial morphology in autistic fibroblasts. An increased expression of the genes that encode for molecules involved in mitochondrial fission/fusion processes, was also detected. In addition, overregulation of PINK1, but decreased Parkin expression, both associated to mitophagy process, were observed. Taken together our findings provide new insights on the possible contribution of the altered mitochondrial quality control pathway in the redox imbalance of ASD patients.