Lipid Peroxidation By-products Research Articles

Dimercaprol is an acrolein scavenger that mitigates acrolein-mediated PC-12 cells toxicity and reduces acrolein in rat following spinal cord injury.

Acrolein is one of the most toxic byproducts of lipid peroxidation, and it has been shown to be associated with multiple pathological processes in trauma and diseases, including spinal cord injury, multiple sclerosis, and Alzheimer's disease. Therefore, suppressing acrolein using acrolein scavengers has been suggested as a novel strategy of neuroprotection. In an effort to identify effective acrolein scavengers, we have confirmed that dimercaprol, which possesses thiol functional groups, could bind and trap acrolein. We demonstrated the reaction between acrolein and dimercaprol in an abiotic condition by nuclear magnetic resonance spectroscopy. Specifically, dimercaprol is able to bind to both the carbon double bond and aldehyde group of acrolein. Its acrolein scavenging capability was further demonstrated by invitro results that showed that dimercaprol could significantly protect PC-12 cells from acrolein-mediated cell death in a dose-dependent manner. Furthermore, dimercaprol, when applied systemically through intraperitoneal injection, could significantly reduce acrolein contents in spinal cord tissue following a spinal cord contusion injury in rats, a condition known to have elevated acrolein concentration. Taken together, dimercaprol may be an effective acrolein scavenger and a viable candidate for acrolein detoxification.

Molecular effects of hesperetin, a citrus flavanone on7,12-dimethylbenz(a)anthracene induced buccal pouch squamous cell carcinoma in golden Syrian hamsters

In recent years, researchers have been focused on citrus flavanone, a naturally occurring bioactive substance of hesperetin. To investigate the molecular mechanism based chemopreventive efficacy of hesperetin on 7,12-dimethylbenz(a)anthracene (DMBA) induced hamster buccal pouch (HBP) squamous cell carcinoma (SCC). The oral tumour was provoked by painted with 0.5% DMBA on left buccal pouch thrice a week for 10 consecutive weeks developed well-differentiated SCC and tumour formation was 100% in DMBA alone. We evaluated the chemopreventive potential of hesperetin by assessing the lipid peroxidation (LPO) by-products, status of enzymatic, non-enzymatic antioxidants, detoxifying agents etc. Moreover, modulating expression of apoptotic and cell proliferation markers were observed in HBP SCC experimental hamsters. Oral administration of hesperetin (20 mg/kg b.w.) to DMBA painted hamsters significantly reversed the stages of oral SCC. Our findings indicate that hesperetin possesses a chemopreventive effect in DMBA-induced oral SCC by exerting anti-carcinogenic property.

Susceptibility of Stallion Spermatozoa to Different Oxidative Challenges: Role of Seminal Plasma

Seminal cryopreservation provides several advantages in horse breeding. However, improvement on post-thaw sperm survival is still necessary. One of the main factors known to impair post-thaw quality of stallion's sperm is the oxidative damage caused by reactive oxygen species (ROS). In this context, the aim of this study was evaluated the effects of ROS on stallion sperm and the possible influence of seminal plasma (SP). Toward this aim, 13 ejaculates from adult stallions (n = 13) were divided into two aliquots which were centrifuged (600g/10 minutes), and the SP was removed and reserved. Pellets were suspended in physiological saline solution (A) or SP (B). Each of these solutions was divided into four aliquots and subjected to challenge with different ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical [OH−]) and the toxic by-product of lipid peroxidation (malondialdehyde [MDA]). Samples were then evaluated for the susceptibility to lipid peroxidation (thiobarbituric acid reactive substances) plasma membrane integrity (eosin–nigrosin), acrosome integrity (Fast Green/rose bengal), mitochondrial activity (3′3 diaminobenzidine), and DNA integrity. Samples incubated in the presence of SP were highly impaired by OH− with regard to motility, plasma membrane integrity, mitochondrial activity, and DNA integrity. On the other hand, in the absence of SP, MDA was highly deleterious, especially with regard to motility, plasma membrane integrity, and mitochondrial activity. Thus, these results indicate that SP may have an important role on the protection of stallion sperm against the damages caused by MDA, an important product of lipid peroxidation.

Bioactivity of arid region honey: an in vitro study

BackgroundAntioxidant and anti-inflammatory properties of honey have been largely recognized by various studies. Almost all of the potential benefits are associated with polyphenol content. Honey varieties from the arid region are reported to be rich in polyphenols, but data related to its bioactivity in vitro is greatly lacking. This study aimed at establishing the antioxidant and anti-inflammatory properties of arid region honey. Four honey varieties from arid region (H1, H2, H3, and H4) and two popular non-arid region honey (H5 and H6) were tested in vitro in this study.MethodsThe erythrocyte membrane protection effect of honey varieties were measured by hemolysis assay after exposing erythrocytes to a peroxide generator. The subsequent production of MDA (malondialdehyde) content in erythrocytes was measured. Immunomodulatory effect of the honey varieties was tested in prostate cancer cells PC-3 and PBMC (peripheral blood mononuclear cells) by measuring the IL-6 (interleukin 6) and NO (nitric oxide) levels in cell culture supernatant after incubation with the honey varieties. PC-3 cell viability was assessed after incubation with honey varieties for 24 h.ResultsArid region honey exhibited superior erythrocyte membrane protection effect with H4 measuring 1.3 ± 0.042mMTE/g and H2 measuring 1.122 ± 0.018mMTE/g. MDA levels were significantly reduced by honey samples, especially H4 (20.819 ± 0.63 nmol/mg protein). We observed a significant decrease in cell population in PC-3 after 24 h in culture on treatment with honey. A moderate increase in NO levels was observed in both cultures after 24 h at the same time levels of IL-6 were remarkably reduced by honey varieties.ConclusionThe results demonstrate the antioxidant effect of arid region honey due to its erythrocyte membrane protection effect and subsequent lowering of oxidative damage as evident from lower levels of lipid peroxidation byproduct MDA. Arid region honey varieties were as good as non-arid region types at decreasing cell viability of prostate cancer cells. The moderate increase in NO levels in PC-3 and PBMCs were not significant enough to elicit any pro-inflammatory response. However, IL-6 secretion was remarkably reduced by all honey varieties in a comparable level indicating the potential anti-inflammatory property of arid region honey.

The role of acetone in the [omim][BF4]-mediated adverse effects on tissues of mussels, human lymphocytes and the fruit fly Drosophila melanogaster

The present study investigated [omim][BF4]-mediated adverse effects on biological models widely used in toxicological studies. Specifically, mussels of the genus Mytilus, human lymphocytes and fruit flies of the species Drosophila melanogaster, were exposed to [omim][BF4] at concentrations ranging from micro- to milligrams per liter, with or without the presence of acetone as a carrier solvent and thereafter [omim][BF4]-mediated adverse effects were analyzed appropriately (stress indices, such as lipid peroxidation byproducts, acetylcholinesterase/AChE activity and micronucleus/MN formation frequency, in mussel gills, Cytokinesis Block Micronucleus/CBMN assay and SMART test in human lymphocytes and fruit flies respectively). LC–MS–TOF analysis was also performed for elucidating [omim][BF4] mode of action in the presence of the carrier solvent. The results showed the toxic potential of [omim][BF4], as well as acetone’s ability to attenuate [omim][BF4]-mediated toxicity in almost all cases, probably due to the significant effect of acetone on the hydrophilic-lipophilic character and the viscosity of [omim][BF4], as well as its interaction and permeability on the cell membranes. The slight involvement of acetone in the attenuation of [omim][BF4]-mediated genotoxic effects on D. melanogaster could be due to species feeding experimental conditions, thus favoring the induction of antioxidant defense system against the [omim][BF4]-mediated effects in all cases.

Analysis of Oxidative Stress in Chronic Exposure to Petroleum Hydrocarbons in Karnataka, India

Background:Several studies have reported the toxicological implications of inhalation of petroleum hydrocarbon fumes in animal models. But, there is certainly little or no documentation of the exposure to petroleum hydrocarbon fuel on oxidative stress levels in humans, unlike the pulmonary physiology. The present study was carried out to evaluate the effects of constituents of the hydrocarbon fuels on oxidative stress levels of the petrol fillers and tanker drivers. Methods: The study involved 165 males divided into three groups were the petrol fillers, tanker drivers and the controls. Case control data set was established wherein the control subjects are not exposed to hydrocarbon fuels with similar age. Serum samples of the subjects were collected and subjected for various biochemical assays. The enzymatic antioxidants such as superoxide dismutase, malondialdehyde a byproduct of lipid peroxidation and total antioxidant capacity of the individuals along with non-enzymatic antioxidant Vitamin A was estimated. Results: The results showed a no significant differences for age, body mass index, superoxide dismutase and levels of Malondialdehyde and total antioxidant capacity. But on the other hand, there is significant changes observed for total antioxidant capacity and vitamin A when exposed group is compared with control subject. Conclusion: It is evidential from the present study that prolonged exposure to petroleum hydrocarbon fumes leads to an increase in their oxidative stress in turn resulting broad spectrum of diseases. Hence, there is a raised need for public awareness about the health hazards in order to enable petrol attendants.

Nitration of tyrosines in complement factor H domains alters its immunological activity and mediates a pathogenic role in age related macular degeneration.

Nitrosative stress has been implicated in the pathogenesis of age related macular degeneration (AMD). Tyrosine nitration is a unique type of post translational modification that occurs in the setting of inflammation and nitrosative stress. To date, the significance and functional implications of tyrosine nitration of complement factor H (CFH), a key complement regulator in the eye has not been explored, and is examined in this study in the context of AMD pathogenesis.Sections of eyes from deceased individuals with AMD (n = 5) demonstrated the presence of immunoreactive nitrotyrosine CFH. We purified nitrated CFH from retinae from 2 AMD patients. Mass spectrometry of CFH isolated from AMD eyes revealed nitrated residues in domains critical for binding to heparan sulphate glycosaminoglycans (GAGs), lipid peroxidation by-products and complement (C) 3b.Functional studies revealed that nitrated CFH did not bind to lipid peroxidation products, nor to the GAG of perlecan nor to C3b. There was loss of cofactor activity for Factor I mediated cleavage of C3b with nitrated CFH compared to non-nitrated CFH. CFH inhibits, but nitrated CFH significantly potentiates, the secretion of the pro-inflammatory and angiogenic cytokine IL-8 from monocytes that have been stimulated with lipid peroxidation by-products. AMD patients (n = 30) and controls (n = 30) were used to measure plasma nitrated CFH using a novel ELISA. AMD patients had significantly elevated nitrated CFH levels compared to controls (p = 0.0117). These findings strongly suggest that nitrated CFH contributes to AMD progression, and is a target for therapeutic intervention.

Assessment of Lipid Peroxidation and Antioxidant Status in Vanillic Acid Treated 7,12-Dimethylbenz[a]anthracene Induced Hamster Buccal Pouch Carcinogenesis.

Vanillic acid, a naturally occurring bioactive substance, possesses diverse pharmacological potential including free radical scavenging and anticancer properties. Excessive generation of Reactive Oxygen Species (ROS) and insufficient antioxidant potential has been involved in numerous pathological disorders, including cancer. To explore the anti-lipid peroxidative and antioxidant efficacy of vanillic acid in Dimethylbenz[a]Anthracene (DMBA) induced oral carcinogenesis. Topical application of DMBA for 14 weeks in the buccal pouch of hamsters resulted in well developed oral squamous cell carcinoma. Vanillic acid at a dosage of 200 mg/kg body weight was orally administrated to the hamsters for 14 weeks. The status of lipid peroxidation and antioxidants were measured in the plasma and buccal mucosa of hamsters using specific colorimetric methods. Altered levels of lipid peroxidation by-products {Thiobarbituric Acid Reactive Substances (TBARS)} and disturbances in antioxidants status {Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), vitamin E, vitamin C and reduced Glutathione (GSH)} were observed in the plasma and buccal mucosa tissues of hamsters treated with DMBA alone. Vanillic acid (200 mg/kg bw p.o) significantly restored the above mentioned plasma and buccal mucosa biochemical variables to near normal range in DMBA treated hamsters. Present findings thus confirm the anti-lipid peroxidative and antioxidant efficacy of vanillic acid in DMBA induced hamster buccal pouch carcinogenesis.

PH modulation ameliorates the red blood cell storage lesion in a murine model of transfusion

BackgroundProlonged storage of packed red blood cells (pRBCs) induces a series of harmful biochemical and metabolic changes known as the RBC storage lesion. RBCs are currently stored in an acidic storage solution, but the effect of pH on the RBC storage lesion is unknown. We investigated the effect of modulation of storage pH on the RBC storage lesion and on erythrocyte survival after transfusion. MethodsMurine pRBCs were stored in Additive Solution-3 (AS3) under standard conditions (pH, 5.8), acidic AS3 (pH, 4.5), or alkalinized AS3 (pH, 8.5). pRBC units were analyzed at the end of the storage period. Several components of the storage lesion were measured, including cell-free hemoglobin, microparticle production, phosphatidylserine externalization, lactate accumulation, and byproducts of lipid peroxidation. Carboxyfluorescein-labeled erythrocytes were transfused into healthy mice to determine cell survival. ResultsCompared with pRBCs stored in standard AS3, those stored in alkaline solution exhibited decreased hemolysis, phosphatidylserine externalization, microparticle production, and lipid peroxidation. Lactate levels were greater after storage in alkaline conditions, suggesting that these pRBCs remained more metabolically viable. Storage in acidic AS3 accelerated erythrocyte deterioration. Compared with standard AS3 storage, circulating half-life of cells was increased by alkaline storage but decreased in acidic conditions. ConclusionsStorage pH significantly affects the quality of stored RBCs and cell survival after transfusion. Current erythrocyte storage solutions may benefit from refinements in pH levels.

EFFECTS OF CAPSAICIN SUPPLEMENTATION ON PRODUCTIVE AND PHYSIOLOGICAL PERFORMANCE OF PEKIN DUCKS DURING SUMMER SEASON

The aim of this study was to evaluate the effect of using graded levels of capsaicin on growth performance, nutrients digestibility coefficients, serum metabolites, oxidative responses and intestinal digestive enzymes activity of Pekin ducks. One hundred and twenty 14d old Pekin ducks were randomly divided into four groups of 30 ducks. The first group was fed on the basal diet (control), while groups 2, 3 and 4 were given the basal diet supplemented with 50, 100 and 150 ppm capsaicin, respectively. The results showed significant improvement in body weight and feed conversion of ducks, particularly with the high level (150ppm) followed by those of the mid one (100pm). Moreover, supplemental capsaicin with different levels enhanced digestibility coefficient values especially with ether extract (EE) and nitrogen free extract (NFE) in Pekin ducks. The serum level of total proteins, globulin, high density lipoprotein (HDL), triiodothyronine (T3), thyroxin (T4) and antioxidant enzymes was significantly increased for ducks fed on diets contained either 100 or 150ppm capsaicin. But, the concentrations of total lipid, cholesterol, triglycerides, low density lipoprotein (LDL), glucose and malondialdehyde (MDA), which is the primary stable by-product of lipid peroxidation, were reduced. On the other hand, the albumin level didn’t significantly affected by dietary capsaicin supplementation. A significant increase in the activities of amylase, lipase and trypsin enzymes was found throughout the small intestine portions with supplemental capsaicin. It is concluded that, capsaicin supplementation with 100ppm up to 150ppm was sufficient to enhance the growth performance traits, nutrients digestibility, thyroid and antioxidant system in growing Pekin ducks. In addition to, using these natural feed additives had clear favorable effect on enzymatic and microbiological profile in small intestine, without adverse effects on liver activity.

Correlation between Endothelin-1 and oxidative stress in apparently healthy obese men

Background and objective: Obesity is the risk for many disease conditions and is becoming a global issue. Endothelial dysfunction and oxidative stress are linked to obesity. Endothelin-1 is a useful marker of endothelial dysfunction. Oxidative stress can be assessed by estimation of lipid peroxidation byproduct malondialdehyde. In this study, we tried to find out the correlation between Endothelin-1 and oxidative stress among apparently healthy obese men. Methods: Eighty apparently healthy non-smoker adult men have participated in this study. Their age ranged between 18 and 50 years. Their serum Endothelin-1 (ELISA), malondialdehyde and lipid parameters (biochemical assays) have been estimated. Results: Subjects were divided into three groups: normal weight (n = 25), overweight (n = 34) and obese (n = 26) basing on their body mass index. The study shows that Endothelin-1 is significantly correlated with each of Malondialdehyde and body mass index (r = 0.262, P = 0.019; r = 0.65, P <0.001 respectively). Malondialdehyde is also significantly positively correlated with BMI (r = 0.378, P = 0.001). Conclusion: Endothelial dysfunction and oxidative stress coexist in obesity. But which of them precedes the other? Is still a question needs to be further studied

Urinary 3-hydroxypropyl mercapturic acid (3-HPMA) concentrations in dogs with acute spinal cord injury due to intervertebral disc herniation

The aim of this study was to investigate urinary 3-hydroxypropyl mercapturic acid (3-HPMA), a metabolite of acrolein, as a novel biomarker in acute spinal cord injury (ASCI) due to intervertebral disc herniation in dogs. Urine from 10 client-owned dogs with ASCI collected at presentation and 10 control dogs was analyzed for 3-HPMA. The median urinary 3-HPMA concentration in ASCI dogs was significantly higher than in control dogs, but was not correlated with the severity of ASCI. The median urinary 3-HPMA concentration in intact dogs was higher than in neutered dogs. Higher urinary 3-HPMA concentrations in dogs after ASCI support a role for acrolein, a cytotoxic by-product of lipid peroxidation, in canine ASCI. Urinary 3-HPMA could be used as a biomarker in future clinical trials to measure the effect of therapeutic intervention of reducing acrolein after ASCI.

Therapeutic relevance of mTOR inhibition in murine succinate semialdehyde dehydrogenase deficiency (SSADHD), a disorder of GABA metabolism

Aldehyde dehydrogenase 5a1-deficient (aldh5a1−/−) mice, the murine orthologue of human succinic semialdehyde dehydrogenase deficiency (SSADHD), manifest increased GABA (4-aminobutyric acid) that disrupts autophagy, increases mitochondria number, and induces oxidative stress, all mitigated with the mTOR (mechanistic target of rapamycin) inhibitor rapamycin [1]. Because GABA regulates mTOR, we tested the hypothesis that aldh5a1−/− mice would show altered levels of mRNA for genes associated with mTOR signaling and oxidative stress that could be mitigated by inhibiting mTOR. We observed that multiple metabolites associated with GABA metabolism (γ-hydroxybutyrate, succinic semialdehyde, D-2-hydroxyglutarate, 4,5-dihydrohexanoate) and oxidative stress were significantly increased in multiple tissues derived from aldh5a1−/− mice. These metabolic perturbations were associated with decreased levels of reduced glutathione (GSH) in brain and liver of aldh5a1−/− mice, as well as increased levels of adducts of the lipid peroxidation by-product, 4-hydroxy-2-nonenal (4-HNE). Decreased liver mRNA levels for multiple genes associated with mTOR signaling and oxidative stress parameters were detected in aldh5a1−/− mice, and several were significantly improved with the administration of mTOR inhibitors (Torin 1/Torin 2). Western blot analysis of selected proteins corresponding to oxidative stress transcripts (glutathione transferase, superoxide dismutase, peroxiredoxin 1) confirmed gene expression findings. Our data provide additional preclinical evidence for the potential therapeutic efficacy of mTOR inhibitors in SSADHD.

4-Hydroxynonenal dependent alteration of TRPV1-mediated coronary microvascular signaling

We demonstrated previously that TRPV1-dependent regulation of coronary blood flow (CBF) is disrupted in diabetes. Further, we have shown that endothelial TRPV1 is differentially regulated, ultimately leading to the inactivation of TRPV1, when exposed to a prolonged pathophysiological oxidative environment. This environment has been shown to increase lipid peroxidation byproducts including 4-Hydroxynonenal (4-HNE). 4-HNE is notorious for producing protein post-translation modification (PTM) via reactions with the amino acids: cysteine, histidine and lysine. Thus, we sought to determine if 4-HNE mediated post-translational modification of TRPV1 could account for dysfunctional TRPV1-mediated signaling observed in diabetes. Our initial studies demonstrate 4-HNE infusion decreases TRPV1-dependent coronary blood flow in C57BKS/J (WT) mice. Further, we found that TRPV1-dependent vasorelaxation was suppressed after 4-HNE treatment in isolated mouse coronary arterioles. Moreover, we demonstrate 4-HNE significantly inhibited TRPV1 currents and Ca2+ entry utilizing patch-clamp electrophysiology and calcium imaging respectively. Using molecular modeling, we identified potential pore cysteines residues that, when mutated, could restore TRPV1 function in the presence of 4-HNE. Specifically, complete rescue of capsaicin-mediated activation of TRPV1 was obtained following mutation of pore Cysteine 621. Finally, His tag pull-down of TRPV1 in HEK cells treated with 4-HNE demonstrated a significant increase in 4-HNE binding to TRPV1, which was reduced in the TRPV1 C621G mutant. Taken together these data suggest that 4-HNE decreases TRPV1-mediated responses, at both the in vivo and in vitro levels and this dysfunction can be rescued via mutation of the pore Cysteine 621. Our results show the first evidence of an amino acid specific modification of TRPV1 by 4-HNE suggesting this 4-HNE-dependent modification of TRPV1 may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.

Abstract 1713: Macrophage-induced bystander effect activates Wnt/β-catenin signaling and induces cellular dedifferentiation

Abstract Cancer stem cells (CSCs) in colorectal cancer (CRC) help maintain tumor heterogeneity, promote tumor growth, invasion, and metastasis, and produce resistance to therapeutic agents. The origin of colorectal CSCs, however, is unclear. Our recent studies showed that a macrophage-induced bystander effect (MIBE) induced gene expression of stem/progenitor cell markers including Ly6A/E and Dclk1 in murine colonic epithelial cells, implying that MIBE helps induce colorectal CSCs. In current studies we show that commensal-triggered MIBE activates Wnt/β-catenin signaling and induces dedifferentiation of colonic epithelial cells during colorectal carcinogenesis, thus contributing to the origin of colorectal CSCs. Exposure of murine primary epithelial cells (YAMC) to commensal-polarized macrophages induced phosphorylated Gsk3β (Ser9), active β-catenin, and Tcf4, suggesting activation of Wnt/β-catenin signaling by MIBE. In vivo, active β-catenin was evident in both stromal and epithelial cells for colon biopsies from E. faecalis-colonized Il10−/− mice compared to sham-colonized mice. In addition, we showed that 4-hydroxynonenal (4-HNE), a byproduct of lipid peroxidation of ω6 polyunsaturated fatty acids, and tumor necrosis factor α (TNFα) mediated activation of Wnt/β-catenin signaling. Epigenetic analysis revealed that MIBE activated Wnt/β-catenin signaling through DNA methylation in the CpG islands of secreted frizzled-related protein 2 (Sfrp2), a soluble modulator of Wnt. Next, we showed that exposure of YAMC cells to 4-HNE and TNFα induced expression of c-Myc, Klf4, Oct4, and Sox2―factors essential for pluripotent stem cells. Immunofluorescent staining of colon biopsies from E. faecalis-colonized Il10−/− mice showed increased expression of these factors in colonic epithelial cells, suggesting dedifferentiation induced by MIBE. This was confirmed by qRT-PCR and Western blots that showed increased expression of Dclk1 and CD44, two markers for colorectal CSCs. Furthermore, increased expression of DCLK1 was noted in human tubular adenomas and invasive CRCs compared to hyperplastic polyps. Finally, inhibition of β-catenin/TCF4 using FH535, and by silencing CTNNB1, decreased DCLK1 expression in HCT116 human colon cancer cells, supporting the notion that Wnt/β-catenin signaling induces cellular dedifferentiation leading to colorectal CSCs. In summary, these results demonstrate that commensal-polarized macrophages activate Wnt/β-catenin signaling and induce dedifferentiation of colonic epithelial cells through 4-HNE and TNFα-mediated bystander effects. These findings provide new evidence for the origin of CSCs in colorectal carcinogenesis and should lead to novel strategies for CRC prevention and therapy. Citation Format: Xingmin Wang, Yonghong Yang, Mark M. Huycke. Macrophage-induced bystander effect activates Wnt/β-catenin signaling and induces cellular dedifferentiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1713.

Loach embryos prooxidant-antioxidant status under the influence of amide derivatives of 1,4-naphthoquinone.

The mechanisms of disorders in cell functions induced by 1,4-naphthoquinone amide derivatives are not clarified yet. The article is dedicated to the study of features of these substances influence on loach Misgurnus fossilis L. embryos pro/antioxidant homeostasis during early embryogenesis. The aim of this work was to study the effect of 2-chloro-3-hydroxy-1,4-naphthoquinone, 2-chloro-3-(3-oxo-3-(piperidine-1-yl)propylamine)-1,4-naphthoquinone (FO-1), 2-chloro-3-(3-(morpholine-4-yl)-3-oxopropylamine)-1,4-naphthoquinone (FO-2 at concentrations of 10-3, 10-5, 10-7 M on the content of TBA-reactive substances (a byproduct of lipid peroxidation) and the activities of superoxide dismutase and catalase in loach embryos. It was established that 1,4-naphthoquinone amide derivatives and 2-chloro-3-hydroxy-1,4-naphthoquinone decreased the content of lipid peroxidation products in embryo cells in a dose-dependent manner. The investigated compounds cause an increase in superoxide dismutase and catalase activities compared with the control value. The results of the two-factor ANOVA test indicate that 2-chloro-3-hydroxy-1,4-naphthoquinone and 1,4-naphthoquinone amide derivatives (FO-1, FO-2) have predominant influence on the TBA-reactive substances content and superoxide dismutase activity. However, the time of loach embryos development has a more pronounced effect on catalase activity than the studied 1,4-naphthoquinone derivatives.

Preliminary case control study to establish the correlation between novel peroxidation biomarkers in cord serum and the severity of hypoxic ischemic encephalopathy

BackgroundHypoxic-ischemic encephalopathy (HIE) has deleterious neurological consequences. To identify patients at risk of neuronal damage deserving implementation of neuroprotective strategies clinicians have relied on prenatal sentinel events, postnatal clinical assessment (Apgar score), and blood gas analysis.This feasibility study aimed to assess if lipid peroxidation byproducts associated with neuronal damage correlated with cord blood metabolic acidemia in patients with HIE.Population and Methods: This is a case/control study in which cases were newborn infants with severe acidemia (pH<7.00; base excess ≥12mmol/L) while control babies exhibited normal gases (pH=7.20–7.40; base excess=−4 to +4mmol/L) in the first cord blood analysis performed immediately after birth. Concomitantly, lipid peroxidation byproducts were determined using ultra performance liquid chromatography coupled to mass spectrometry in the same cord blood sample. ResultsA total of 19 controls and 20 cases were recruited. No differences in gestational characteristics were present. However, cases exhibited profound metabolic alterations as compared to controls (Cases vs. Control: pH=6.90±0.1 vs. 7.33±0.03; base excess=−15±3 vs. −1±2mmol/L), 85% were admitted to the NICU, and 50% developed symptoms of HIE. 8-iso-15(R)-PGF2α (P=0.01) and total isoprostanes (P=0.045) presented statistically significant differences between cases and control groups and correlated with level of HIE. ConclusionsThe 8-iso-15(R)-PGF2α and isoprostanes reflecting oxidative damage are significantly increased in severe postnatal acidemia. Follow up studies with adequate power are necessary to confirm if these biomarkers measured in cord blood serum could be predictive of neonatal encephalopathy.

Effect of malonaldehyde cross-linking on the ability of shrimp tropomyosin to elicit the release of inflammatory mediators and cytokines from activated RBL-2H3 cells.

Malonaldehyde, the primary by-product of lipid peroxidation in food, modifies the structural and functional properties of proteins by cross-linking. The aim of this study was to investigate the effect of malonaldehyde on the allergenicity of shrimp tropomyosin. RBL-2H3 cells, a model of type I allergic reactions, were sensitised with sera from patients allergic to shrimp, and were stimulated with native and cross-linked tropomyosin. Release of inflammatory mediators such as β-hexosaminidase, histamine, tryptase, cysteinyl leukotriene, and prostaglandin D2 was clearly suppressed in a manner that depended on the extent of tropomyosin cross-linking. Release of interleukin-4 (IL-4) and IL-13 was similarly decreased. Notably, cells sensitised with one patient's serum released IL-4 at comparable levels in response to native and cross-linked tropomyosin. Cross-linking strongly modulates the ability of shrimp tropomyosin to induce release of inflammatory cytokines and mediators from activated RBL-2H3 cells. © 2016 Society of Chemical Industry.

Comprehensive Assessment of Oxidatively Induced Modifications of DNA in a Rat Model of Human Wilson's Disease

Defective copper excretion from hepatocytes in Wilson's disease causes accumulation of copper ions with increased generation of reactive oxygen species via the Fenton-type reaction. Here we developed a nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry coupled with the isotope-dilution method for the simultaneous quantification of oxidatively induced DNA modifications. This method enabled measurement, in microgram quantities of DNA, of four oxidative stress-induced lesions, including direct ROS-induced purine cyclonucleosides (cPus) and two exocyclic adducts induced by byproducts of lipid peroxidation, i.e. 1,N(6)-etheno-2'-deoxyadenosine (εdA) and 1,N(2)-etheno-2'-deoxyguanosine (εdG). Analysis of liver tissues of Long-Evans Cinnamon rats, which constitute an animal model of human Wilson's disease, and their healthy counterparts [i.e. Long-Evans Agouti rats] showed significantly higher levels of all four DNA lesions in Long-Evans Cinnamon than Long-Evans Agouti rats. Moreover, cPus were present at much higher levels than εdA and εdG lesions. In contrast, the level of 5-hydroxymethyl-2'-deoxycytidine (5-HmdC), an oxidation product of 5-methyl-2'-deoxycytidine (5-mdC), was markedly lower in the liver tissues of Long-Evans Cinnamon than Long-Evans Agouti rats, though no differences were observed for the levels of 5-mdC. In vitro biochemical assay showed that Cu(2+) ions could directly inhibit the activity of Tet enzymes. Together, these results suggest that aberrant copper accumulation may perturb genomic stability by elevating oxidatively induced DNA lesions, and by altering epigenetic pathways of gene regulation.

Abstract WP103: Mitochondrial/endoplasmic Reticulum Dysfunction Exacerbates Oxidative Damage in Aging White Matter Ischemia

White matter (WM) is frequently affected by stroke. Aging WM recovers less following stroke compared to younger WM. Free radicals accumulate with aging resulting in increased oxidative stress. Mitochondria/endoplasmic reticulum (ER) interactions are crucial for protection from oxidative stress. We hypothesized that aging WM is more susceptible to oxidative stress damage mediated by mitochondrial/ER dysfunction, leading to decreased ATP synthesis and impaired Ca2+ homeostasis. Mouse optic nerves (MON) obtained from C57BL/6J and Thy-1 CFP(+) mice at 1 (young) or 12 months (aging) of age were used. Axon function was quantified by recording evoked compound action potentials. Mitochondrial/ER structure and interactions were determined by 3D-electron microscopy, Western blotting, ATP assays, and CFP (+) fluorescent mitochondrial imaging. Oxidative stress was assessed by NOS and glutathione assays and by Western blotting for oxidative stress markers, 3-NT and 4-HNE. Oxygen-glucose deprivation (OGD) for 60 min resulted in decreased axon function recovery of aging WM. Aging WM showed elevated NOS activity and increased levels of by-products of lipid peroxidation (4-HNE) and protein nitration (3-NT), indicating aggravated oxidative stress. Structurally, aging axons were larger, with thicker myelin, and were characterized by longer and thicker mitochondria due to altered levels of mitochondrial shaping proteins. This was further confirmed by 3D-EM and CFP (+) imaging and may underlie the decreased ATP levels detected in aging WM. Moreover, mitochondrial-ER interactions were compromised due to decreased association between the organelles and due to decrease in levels of mitochondrial trafficking protein miro-2, which suggests defective Ca2+ homeostasis in aging axons. Calnexin, an ER stress response chaperone protein, was decreased under baseline conditions in aging axons, but did not show upregulation following OGD when compared to young axons, suggesting a defect in unfolded protein responses. We conclude that aging WM is increasingly vulnerable to stroke because of inherent structural changes in axons and mitochondria/ER interactions, leading to depletion of ATP and defective Ca2+ dynamics, resulting in increased oxidative stress.