Endogenous Lipid Peroxidation Research Articles

Enhancing lipid peroxidation via radical chain transfer reaction for MRI guided and effective cancer therapy in mice

Lipid peroxidation (LPO), the process of membrane lipid oxidation, is a potential new form of cell death for cancer treatment. However, the radical chain reaction involved in LPO is comprised of the initiation, propagation (the slowest step), and termination stages, limiting its effectiveness in vivo. To address this limitation, we introduce the radical chain transfer reaction into the LPO process to target the propagation step and overcome the sluggish rate of lipid peroxidation, thereby promoting endogenous lipid peroxidation and enhancing therapeutic outcomes. Firstly, radical chain transfer agent (CTA-1)/Fe nanoparticles (CTA-Fe NPs-1) was synthesized. Notably, CTA-1 convert low activity peroxyl radicals (ROO·) into high activity alkoxyl radicals (RO·), creating the cycle of free radical oxidation and increasing the propagation of lipid peroxidation. Additionally, CTA-1/Fe ions enhance reactive oxygen species (ROS) generation, consume glutathione (GSH), and thereby inactivate GPX-4, promoting the initiation stage and reducing termination of free radical reaction. CTA-Fe NPs-1 induce a higher level of peroxidation of polyunsaturated fatty acids in lipid membranes, leading to highly effective treatment in cancer cells. In addition, CTA-Fe NPs-1 could be enriched in tumors inducing potent tumor inhibition and exhibit activatable T1-MRI contrast of magnetic resonance imaging (MRI). In summary, CTA-Fe NPs-1 can enhance intracellular lipid peroxidation by accelerating initiation, propagation, and inhibiting termination step, promoting the cycle of free radical reaction, resulting in effective anticancer outcomes in tumor-bearing mice.

Response of the antioxidant system of wheat seedlings with different genotypes to exogenous prooxidants: the relationship with resistance to abiotic stressors

Oxidative stress is an important component of heat- and drought-induced damage in plants. However­, information on the relationship between the resistance of cultivated plants with different genotypes to environmental factors and their ability to maintain a pro-/antioxidant balance remains contradictory. This study aimed to compare the growth responses and adaptation ability of the antioxidant system in different wheat cultivars to oxidative stress agents hydrogen peroxide and iron(II) sulfate. Etiolated seedlings of common winter wheat (Triticum aestivum L.) Antonivka and Tobak (heat- and drought-tolerant), and Avgustina and Doskonala (not resistant to heat and drought) were used for the study. Three-day-old etiolated seedlings were exposed to 50 mM H2O2 or 5 mM FeSO4 for one day. It was found that seedlings of Antonivka and Tobak cultivars treated with H2O2 or FeSO4 were able to maintain relatively intensive growth, accumulated significantly lower amounts of endogenous hydrogen peroxide and lipid peroxidation products, significantly increased anthocyanin content and had a higher activity of SOD and catalase as compared with non-resistant cultivars. The non-tolerant cultivars’ response to stress agents was only to increase proline content with a simultaneous decrease in SOD activity and anthocyanins content. The identified varietal markers of the antioxidant system adaptive strategy can be used to develop new approaches for screening wheat cultivars with cross-resistance to major abiotic stressors. Keywords: antioxidant system, drought resistan­ce, ferrous sulphate, H2O2, heat tolerance, oxidative stress, Triticum aestivum seedlings

Hepatic Anti-Oxidative Genes CAT and GPX4 Are Epigenetically Modulated by RORγ/NRF2 in Alphacoronavirus-Exposed Piglets.

As a member of alpha-coronaviruses, PEDV could lead to severe diarrhea and dehydration in newborn piglets. Given that lipid peroxides in the liver are key mediators of cell proliferation and death, the role and regulation of endogenous lipid peroxide metabolism in response to coronavirus infection need to be illuminated. The enzymatic activities of SOD, CAT, mitochondrial complex-I, complex-III, and complex-V, along with the glutathione and ATP contents, were significantly decreased in the liver of PEDV piglets. In contrast, the lipid peroxidation biomarkers, malondialdehyde, and ROS were markedly elevated. Moreover, we found that the peroxisome metabolism was inhibited by the PEDV infection using transcriptome analysis. These down-regulated anti-oxidative genes, including GPX4, CAT, SOD1, SOD2, GCLC, and SLC7A11, were further validated by qRT-PCR and immunoblotting. Because the nuclear receptor RORγ-driven MVA pathway is critical for LPO, we provided new evidence that RORγ also controlled the genes CAT and GPX4 involved in peroxisome metabolism in the PEDV piglets. We found that RORγ directly binds to these two genes using ChIP-seq and ChIP-qPCR analysis, where PEDV strongly repressed the binding enrichments. The occupancies of histone active marks such as H3K9/27ac and H3K4me1/2, together with active co-factor p300 and polymerase II at the locus of CAT and GPX4, were significantly decreased. Importantly, PEDV infection disrupted the physical association between RORγ and NRF2, facilitating the down-regulation of the CAT and GPX4 genes at the transcriptional levels. RORγ is a potential factor in modulating the CAT and GPX4 gene expressions in the liver of PEDV piglets by interacting with NRF2 and histone modifications.

The Relationship of Disorders in the Hemostasis System with Changes in Homeostasis in Conditions of Progressive Inflammation

The urgency of the problem of acute peritonitis in surgical practice is due to the annual increase in morbidity, a high number of complications, therapeutic complexity and mortality. Materials and methods. The material of the study was 45 patients with acute peritonitis, who were divided into 3 groups depending on the course of pathology after surgery. The first group (n=15) – without complications, the second (n=15) – with wound complications, the third (n=15) – with tertiary peritonitis and sepsis. In addition to standard research methods, a number of laboratory tests are included to determine the severity of intoxication, oxidative activity, the state of the blood coagulation system, and the functional state of the liver. The study period is the 1st, 5th, 10th postoperative day. Results. The results showed that in the early period after surgery in patients with acute peritonitis, the state of hypercoagulemia, inhibition of fibrinolysis correlate with lipid peroxidation, endogenous intoxication, and violation of the functional status of the liver. With effective therapy, during the early postoperative period, the state of the hemostasis system and homeostasis indicators are corrected against the background of restoration of the functional status of the liver. With the aggravation of the phenomena of inflammation in the early postoperative period and the development of complications, there is a progression of changes in the studied indicators of homeostasis and hemostasis with further inhibition of the functional state of the liver. With the development of tertiary peritonitis and sepsis, the hemostasis system is modified in the form of hypocoagulemia and activation of fibrinolysis against the background of a sharp increase in endogenous intoxication, lipid peroxidation and greater inhibition of the functional status of the liver, including from its hemostasis-regulating component. Conclusions. The obtained results indicate the presence of a reliable correlation of disorders in the hemostasis system with changes in homeostasis in conditions of progressive inflammation.

Obesity Is Associated with Increased F2-Isoprostanes and IL-6 in Black Women

Obesity affects over 40% of the adult population and is a major risk factor for morbidity and mortality due to cardiovascular disease. Black women have one of the highest prevalences of obesity, insulin resistance, hypertension, and cardiovascular events in the US. We previously found that free radical-mediated lipid peroxidation contributes to IL-6 production in dendritic cells leading to inflammation and hypertension. Thus, we hypothesized that F2-isoprostanes (F2-IsoPs), products and biomarkers of endogenous lipid peroxidation, contribute to increased inflammation and IL-6 production among obese Black women. We studied a total of 88 obese Black women of age 42.0 ± 9.8 years, weight 102 ± 16 kg, and body mass index (BMI) 37.68 ± 5.08. Systolic and diastolic blood pressure were 124 ± 14/76.2 ± 9.9 mmHg, heart rate was 68.31 ± 10.26 beats/min, and fasting insulin was 15.0 ± 8.7 uU/mL. Plasma F2-IsoPs were measured using gas chromatography/negative ion chemical ionization mass spectrometry (GC/NICI-MS). Plasma cytokines, including IL-6, IL-8, IL-10, IL-1β, TNF-a, and C-reactive proteins were measured using multiplex Luminex technology. Anthropometric measurements were performed using dual-energy X-ray absorptiometry. Using Pearson’s correlation analysis, we found that BMI was positively correlated with plasma F2-IsoPs, while inversely correlated with insulin sensitivity in obese Black women. Further, F2-IsoPs were positively correlated with inflammatory marker IL-6 levels while negatively correlated with anti-inflammatory marker IL-10. In addition, we found that plasma F2-IsoPs levels were significantly associated with reduced insulin sensitivity. These results suggest that F2-IsoPs may be associated with obesity-induced cardiovascular risk in Black women by increasing the production of inflammatory cytokine IL-6 and decreasing the production of anti-inflammatory IL-10.

Mass Spectrometry Analysis of DNA and Protein Adducts as Biomarkers in Human Exposure to Cigarette Smoking: Acrolein as an Example.

Acrolein is a major component in cigarette smoke and a product of endogenous lipid peroxidation. It is difficult to distinguish human exposure to acrolein from exogenous sources versus endogenous causes, as components in cigarette smoke can stimulate lipid peroxidation in vivo. Therefore, analysis of acrolein-induced DNA and protein adducts by the highly accurate, sensitive, and specific mass spectrometry-based methods is vital to estimate the degree of damage by this IARC Group 2A carcinogen. This Perspective reviews the analyses of acrolein-induced DNA and protein adducts in humans by mass spectrometry focusing on samples accessible for biomonitoring, including DNA from leukocytes and oral cells and abundant proteins from blood, i.e., hemoglobin and serum albumin.

Evaluation of oxidative stress markers in ethanol users

Ethanol is a central nervous system depressant that is widely consumed worldwide. When consumed chronically, it may have several consequences to the organism, such as oxidative stress. Ethanol metabolism increases the production of oxidant molecules and its consumption may cause changes in enzymatic and non-enzymatic systems that maintain cellular homeostasis. The activity of endogenous enzymes and lipid peroxidation are altered in alcohol consumers. Therefore, this study aimed to evaluate oxidative stress parameters in ethanol users compared to a control group. For that, the activity of the enzymes superoxide dismutase, catalase, and glutathione peroxidase, the ferric reducing/antioxidant power (FRAP), and malondialdehyde were evaluated. The influence of the amount of ethanol consumed on the analyzed parameters was also verified. The group of alcohol users consisted of 52 volunteers, 85% male and 15% female, with a mean age of 41±13 years. The control group consisted of 50 non-drinkers, 40% male and 60% female, with a mean age of 50±10 years. There was a significant difference in superoxide dismutase (P<0.001) and malondialdehyde (P=0.007) measurements between groups, as both parameters were increased in the group of ethanol users. Because of the higher amount of ethanol consumed, there was an increase of the catalase activity parameters and gradual reduction of FRAP. Thus, the ethanol-consuming participants were most likely under oxidative stress.

Characterization and Quantification of Acrolein-Induced Modifications in Hemoglobin by Mass Spectrometry─Effect of Cigarette Smoking.

Exposure to acrolein, the smallest α, β-unsaturated aldehyde, in humans originates from cigarette smoking and other environmental sources, food cooking, and endogenous lipid peroxidation and metabolism. The protein modification caused by acrolein is associated with various diseases, including cancer, cardiovascular, and neurodegenerative diseases. In this study, acrolein-modified human hemoglobin was reduced by sodium borohydride. Thus, three types of modifications, that is, Schiff base, Michael addition, and formyl-dehydropiperidion adducts, were converted to the corresponding stable adducts, leading to mass increases of 40.0313, 58.0419, and 96.0575 Da, respectively. These stable acrolein-modified hemoglobin peptides were identified by nanoflow liquid chromatography coupled to a high-resolution nanoelectrospray ionization tandem mass spectrometry. Among the 26 different types and sites of modifications, 15 of them showed a dose-dependent increase with increasing concentrations of acrolein. To investigate the role of acrolein-induced modifications in smoking and oral cancer, the 15 dose-responsive acrolein-modified peptides, together with three ethylated peptides previously identified, were quantified in oral cancer patients, healthy smokers, and healthy nonsmokers. The results reveal that the relative extents of the Michael-type adduct at α-Lys-16, α-His-50, and β-Lys-59 are significantly higher in smokers (oral cancer and healthy) than in nonsmokers. Areas under the receiver operating characteristic curve of these peptides range from 0.7500 to 0.9375, indicating the ability to discriminate smokers from nonsmokers. Additionally, these acrolein-modified peptides correlate with three ethylated peptides at the N-termini of α- and β-globin, as well as β-His-77, and with the number of cigarettes smoked per day. Therefore, measuring the reduced Michael adducts at α-Lys-16, α-His-50, and β-Lys-59 of hemoglobin from one drop of blood by this sensitive and specific method may reflect the increase of acrolein exposure due to cigarette smoking.

High-resolution metabolomics of exposure to tobacco smoke during pregnancy and adverse birth outcomes in the Atlanta African American maternal-child cohort

Exposure to tobacco smoke during pregnancy has been associated with a series of adverse reproductive outcomes; however, the underlying molecular mechanisms are not well-established. We conducted an untargeted metabolome-wide association study to identify the metabolic perturbations and molecular mechanisms underlying the association between cotinine, a widely used biomarker of tobacco exposure, and adverse birth outcomes. We collected early and late pregnancy urine samples for cotinine measurement and serum samples for high-resolution metabolomics (HRM) profiling from 105 pregnant women from the Atlanta African American Maternal-Child cohort (2014–2016). Maternal metabolome perturbations mediating prenatal tobacco smoke exposure and adverse birth outcomes were assessed by an untargeted HRM workflow using generalized linear models, followed by pathway enrichment analysis and chemical annotation, with a meet-in-the-middle approach. The median maternal urinary cotinine concentrations were 5.93 μg/g creatinine and 3.69 μg/g creatinine in early and late pregnancy, respectively. In total, 16,481 and 13,043 metabolic features were identified in serum samples at each visit from positive and negative electrospray ionization modes, respectively. Twelve metabolic pathways were found to be associated with both cotinine concentrations and adverse birth outcomes during early and late pregnancy, including tryptophan, histidine, urea cycle, arginine, and proline metabolism. We confirmed 47 metabolites associated with cotinine levels, preterm birth, and shorter gestational age, including glutamate, serine, choline, and taurine, which are closely involved in endogenous inflammation, vascular reactivity, and lipid peroxidation processes. The metabolic perturbations associated with cotinine levels were related to inflammation, oxidative stress, placental vascularization, and insulin action, which could contribute to shorter gestations. The findings will support the further understanding of potential internal responses in association with tobacco smoke exposures, especially among African American women who are disproportionately exposed to high tobacco smoke and experience higher rates of adverse birth outcomes.

Host phospholipid peroxidation fuels ExoU-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology.

Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.

High-resolution metabolomics of exposure to tobacco smoke during pregnancy and adverse birth outcomes in the Atlanta African American Maternal-Child cohort

BACKGROUND AND AIM: Exposure to tobacco smoke during pregnancy has been associated with a series of adverse reproductive outcomes; however, the underlying molecular mechanisms are not well-established. In this analysis, we measured urinary cotinine, a widely used biomarker of tobacco exposure, and conducted high-resolution metabolomics (HRM) to identify biological perturbations associated with prenatal tobacco smoke exposures and adverse birth outcomes. METHODS: We collected early and late pregnancy urine samples for cotinine measurement and serum samples for HRM profiling from 105 pregnant women from the Atlanta African American Maternal-Child cohort (2014-2016). Using an untargeted HRM workflow, we investigated how the biological perturbations in maternal metabolome mediate the association between prenatal smoke exposures and adverse birth outcomes (preterm birth, early term birth vs. full term birth; gestational age at birth) using a meet-in-the-middle approach. RESULTS:The median maternal urinary cotinine level was 4.77 ug/g creatinine, with 29 subjects higher than 100 ug/g creatinine. In total, 16,481 and 13,043 metabolic features were extracted from serum samples using hilic and c18 columns, respectively. Thirteen metabolic pathways were found to be associated with cotinine level and adverse birth outcomes during early and late pregnancy, including tryptophan, histidine, urea cycle, arginine, and proline metabolism. We confirmed 47 metabolites associated with cotinine exposure, preterm birth, and shorter gestational length, which include glutamate, serine, choline, and taurine; these metabolites are closely involved in endogenous inflammation, vascular reactivity, and lipid peroxidation processes. CONCLUSIONS:The metabolic perturbations associated with cotinine exposure were closely connected within a metabolic network related to inflammation, oxidative stress, placental vascularization, and insulin action, which could contribute to shorter gestations. The findings support the future development of targeted interventions to reduce adverse birth outcomes associated with tobacco smoke exposure, especially among African American who are disproportionately exposed to high tobacco smoke and experience higher rates of adverse birth outcomes. KEYWORDS: Tobacco Smoke Exposures, Metabolomics, Birth Outcomes, Preterm Birth

Dual Mechanisms of Cardiac Action Potential Prolongation by 4-Oxo-Nonenal Increasing the Risk of Arrhythmia; Late Na+ Current Induction and hERG K+ Channel Inhibition.

4-Oxo-nonenal (4-ONE) is an endogenous lipid peroxidation product that is more reactive than 4-hydroxy-nonenal (4-HNE). We previously reported the arrhythmic potential of 4-HNE by suppression of cardiac human Ether-a-go-go Related Gene (hERG) K+ channels with prolonged action potential duration (APD) in cardiomyocytes. Here, we illustrate the higher arrhythmic risk of 4-ONE by modulating the cardiac hNaV1.5 channel currents (INaV). Although the peak amplitude of INaV was not significantly changed by 4-ONE up to 10 μM, the rate of INaV inactivation was slowed, and the late Na+ current (INaL) became larger by 10 μM 4-ONE. The chemical modification of specific residues in hNaV1.5 by 4-ONE was identified using MS-fingerprinting analysis. In addition to the changes in INaV, 4-ONE decreased the delayed rectifier K+ channel currents including the hERG current. The L-type Ca2+ channel current was decreased, whereas its inactivation was slowed by 4-ONE. The APD prolongation by 10 μM of 4-ONE was more prominent than that by 100 μM of 4-HNE. In the computational in silico cardiomyocyte simulation analysis, the changes of INaL by 4-ONE significantly exacerbated the risk of arrhythmia exhibited by the TdP marker, qNet. Our study suggests an arrhythmogenic effect of 4-ONE on cardiac ion channels, especially hNaV1.5.

The HIV-1 protease inhibitor Amprenavir targets Leishmania donovani topoisomerase I and induces oxidative stress-mediated programmed cell death.

The global prevalence of HIV is a major challenge for the control of visceral leishmaniasis. Although the effectiveness and usefulness of amprenavir (APV) are well studied in anti-retroviral regimens, very little is known on HIV/VL-co-infected patients. In the present study, we report for the first time the protective efficacy of APV against visceral leishmaniasis by inhibition of DNA Topoisomerase I (LdTOP1LS) and APV-induced downstream pathway in programmed cell death (PCD). During the early phase of activation, reactive oxygen species (ROS) is increased inside the cells, which causes subsequent elevation of lipid peroxidation. Endogenous ROS formation and lipid peroxidation cause eventual depolarization of mitochondrial membrane potential (ΔΨm). Furthermore, the release of cytochrome c and activation of CED3/CPP32 group of proteases lead to the formation of oxidative DNA lesions followed by DNA fragmentation. The promising in vitro and ex vivo results promoted to substantiate further by in vivo animal experiment, which showed a significant reduction of splenic and hepatic parasites burden compared to infected controls. Interestingly, APV selectively targets LdTOPILS and does not inhibit the catalytic activity of human topoisomerase I (hTopI). Moreover, based on the cytotoxicity test APV is not toxic for host macrophage cells, which is correlated with non-responsiveness of inhibition of catalytic activity of hTopI. Taken together, this study provides the opportunity for discovering and evaluating newer potential molecular therapeutic targets for drug designing. The present study might be exploited in future as important therapeutics, which will be useful for treatment of VL as well as HIV-VL co-infection.

Antioxidant, Anti-inflammatory, and Anti-arthritic Effect of Thymoquinone-rich Black Cumin (Nigella sativa) oil (BlaQmax®) on Adjuvant-induced Arthritis

Rheumatoid Arthritis (RA) is a complex autoimmune disorder involving chronic and persistent inflammation, principally influencing the synovial joints which further prompting the obliteration of articular cartilage. Although black cumin (Nigella sativa) oil has already studied for its anti-arthritic properties, the current study was focused on the comparative evaluation of the antioxidant and anti-inflammatory properties of a thymoquinone (TQ)-rich (5% w/v) black cumin oil (BQ) with the commonly available standard black cumin oil (BM) containing 0.4% (w/v) TQ, and subsequent investigation on the potential application of BQ in the management of RA. Adjuvant-induced arthritis (AA) was instigated by a single intradermal infusion of 0.1 mL of Complete Freund&amp;#39;s adjuvant (CFA) on the paw of adult Wistar rats. Based on the primary dose-response study using the carrageenan-induced paw edema model, 50 mg/kg b.wt. of BQ was employed for the treatment. The endogenous antioxidants (SOD, Catalase, GPx, and GSH), pro-inflammatory cytokines (COX-2, Nitrate, iNOS, TNF-&amp;alpha;, IL-6), lipid peroxidation, and histopathology were evaluated to monitor the influence of BQ in AA rats. Adjuvant-induced animals showed a critical downregulation in antioxidant status with elevated levels of pro-inflammatory cytokines and lipid peroxidation. But, the treatment with BQ significantly reversed the antioxidant and inflammatory markers with downregulation of the pro-inflammatory gene expressions. Histopathology showed a significant reduction in the massive cell infiltration and epidermal edema of the paw tissue in AA rats when administered with BQ and indicated its potential effect to alleviate RA conditions in experimental rats.

Hepatic distress syndrome in surgery: concept, pathogenesis, prevention and correction

To establish the universality and regularity of hepatic disorders, their role in pathogenesis of homeostasis impairment and determine scientific base for a new syndrome - hepatic distress syndrome in surgery. Chronic experiments on outbred dogs were carried out. Acute peritonitis was simulated in the first group (n=15), acute destructive pancreatitis in the second group (n=15), acute obstructive intestinal obstruction in the third group (15). In the fourth group, experimental acute peritonitis (n=15) was followed by Remaxol infusions (20 ml/kg). Liver function was evaluated using the indicators of endogenous intoxication, lipid metabolism markers, as well as composition of lipids in liver tissue. Clinical studies included 44 patients with peritonitis (the first subgroup - standard therapy (n=20), Remaxol infusion in the second subgroup (n=24), severe acute pancreatitis (n=18), acute adhesive intestinal obstruction (n=20)). Patients underwent surgery. Along with routine indicators, we analyzed severity of endogenous intoxication, lipid peroxidation, phospholipase activity, serum lipids and red blood cell count. Experimental and clinical studies have shown significant liver damage in all diseases with various pathogenesis. Regardless of urgent disease, one of the leading component is membrane-destabilizing process. The last one is determined by excessive activity of membrane lipid peroxidation and phospholipases in liver tissue. Severe abnormalities are followed by impairment of liver detoxification ability and liver may be a source of toxins per se. Remaxol infusion in the treatment of experimental and clinical acute peritonitis increased liver tolerance to trigger pathogenetic agents. This process resulted improvement of laboratory and clinical parameters. In general, we obtained high significance of liver damage in pathogenesis of acute urgent abdominal pathology. Experimental and clinical data for determining hepatic distress syndrome as important aspect in acute abdominal surgical diseases have been obtained. Hepatic distress syndrome in surgery is a combination of abnormal processes with progression of endogenous intoxication, oxidative stress and phospholipase activity following membrane-destabilizing phenomena and secondary liver dysfunction.

Effects of k-carrageenan supplementation or in combination with cholesterol-loaded cyclodextrin following freezing-thawing process of rooster spermatozoa

This experimental research purposely seeks to explore the effect of supplementing k-carrageenan (k-CRG) or CLC (cholesterol-loaded cyclodextrins) or the combined effect of k-CRG and CLC as supplements of antioxidants to an extender for rooster semen freezing. A total of 75 neat pooled ejaculates were collected twice a week from twenty-five (25) commercial line arbor acres broiler roosters (30 wks) during the experimental period. In each replicate, semen samples (n= 15, three ejaculates per rooster) were pooled and divided into nine equal aliquots, and each aliquot was diluted with one of the following extender supplemented with k-CRG, CLC, and k-CRG + CLC after which it was subjected to cryopreservation process using the “pellet” method. In study I, the supplementation of extenders with k-CRG was in five equal aliquots as follows; (0.2, 0.4, 0.6, 0.8) mg/mL and control group (k-CRG 0) mg/mL while in Study II, there was a combination of both k-CRG + CLC (0.4 mg/mL + 1.5 mg/mL, respectively), 0.4 mg/mL k-CRG, 1.5 mg/mL CLC and control group. Sperm quality parameters, endogenous antioxidant enzymes, lipid peroxidation (MDA) and ROS were all assessed after the freeze-thaw process. Our findings in study I indicated that at post-thaw, an optimum 0.4 mg/mL k-CRG supplementation in the extender improved semen quality parameters, endogenous enzymes, MDA and ROS in comparison to the control group. Interestingly prior to the freeze-thaw process, it was depicted in study II that combined k-CRG + CLC (0.4 mg/mL+1.5 mg/mL) inclusion in the extender provided maximum protection to sperm quality parameters, endogenous enzymes, MDA and ROS in comparison to 1.5 mg/mL CLC and control group at post-thaw. Besides, there was also a significant difference observed in the extenders supplemented with combined k-CRG + CLC (0.4 mg/mL +1.5 mg/mL) when compared to 0.4 mg/mL k-CRG for semen quality parameters and endogenous antioxidant enzymes (SOD, CAT, and GPx) but no significant difference was observed for MDA and ROS. Also, there was a significant difference observed in the extender supplemented with 1.5 mg/mL CLC when compared to the control group for semen quality parameters, SOD, CAT, and MDA but no significant difference for GPx and ROS at post-thaw. In conclusion, k-CRG at an optimal dosage of 0.4 mg/mL proved effective for improving post-thaw sperm quality but its combined addition k-CRG + CLC at an optimal concentration of (0.4 + 1.5) mg/mL in the extender provided greater protection to the rooster spermatozoa at post-thaw.

Genotoxicity of aldehyde mixtures: profile of exocyclic DNA-adducts as a biomarker of exposure to tobacco smoke

Electrophilic compounds present in humans, originating from endogenous processes or pollutant exposures, pose a risk to health though their reaction with nucleophilic sites in protein and DNA. Among this chemical class, aldehydes are mainly present in indoor air and they can also be produced by endogenous lipid peroxidation arising from oxidative stress. Known to be very reactive, aldehydes have the ability to form exocyclic adducts to DNA that, for the most if not repaired correctly, are mutagenic and by consequence potential agents involved in carcinogenesis. The aim of this work was to establish profiles of exocyclic DNA adducts induced by aldehyde mixtures, which could ultimately be considered as a genotoxic marker of endogenous and environmental aldehyde exposure. Adducts were quantified by an accurate, sensitive and validated ultra high performance liquid chromatography-electrospray ionization analytical method coupled to mass spectrometry in the tandem mode (UHPLC-ESI-MS/MS). We simultaneously measured nine exocyclic DNA adducts generated during the exposure in vitro of calf thymus DNA to different concentrations of each aldehyde along, as well as, to an equimolar mixture of these aldehydes. This approach has enabled us to establish dose-response relationships that allowed displaying the specific reactivity of aldehydes towards corresponding adducts formation. Profiles of these adducts determined in DNA of current smokers and non-smokers blood samples supported these findings. These first results are encouraging to explore genotoxicity induced by aldehyde mixtures and can furthermore be used as future reference for adductomic approaches.

Histidine Reactive Chemicals Modify the Thermus thermophilus CuA Redox Center

The electron transport chain is a series of redox protein complexes that couple the shuttling of electrons with the pumping of protons, in order to create the electrochemical gradient that drives ATP synthesis. The first redox center of the terminal complex from the Thermus thermophilus bacteria has been isolated as TtCuA and has a binuclear copper center bridged by two cysteines and ligated by two histidines and two weak axial ligands. H157 of TtCuA, a solvent exposed ligating histidine, has demonstrated modification by diethyl pyrocarbonate (DEPC), an exogenous chemical modifier, that is facilitated by the residue’s fast proton exchange with the solvent. The H157‐DEPC adduct is reversible over 48 hours at room temperature, as supported by mass spectrometry, electrochemistry, and visible CD spectroscopy. This is one of two modifications, out of 12, that becomes removed. Due to the lability of the H157‐DEPC bond, it is possible that the H157 Nɛ is primed for proton pumping in the electron transport chain.In order to model a possible in vivo modification, TtCuA was exposed to 4‐hydroxynonenal (HNE), an endogenous lipid peroxidation product associated with multiple metabolic and neurodegenerative diseases. Exposure of TtCuA to 20 equivalents of HNE appears to reduce the protein over 48 hours and four modifications are observed via mass spectrometry. Exposure of the Pseudomonas aeruginosa azurin protein, a mononuclear blue‐copper denitrification protein with similar ligand groups to TtCuA, is not reduced by HNE, despite modification. Azurin also lacks a ligating histidine that can be modified by DEPC. In conclusion, the binuclear copper center of TtCuA has hyperreactivity in comparison to a similarly ligated mononuclear copper center.Support or Funding InformationMurchison Undergraduate Research Fellowship, Trinity University Chemistry Department, Mach Research Fellowship, Welch Foundation

Redox Signaling via Lipid Peroxidation Regulates Retinal Progenitor Cell Differentiation

Reactive oxygen species (ROS) and downstream products of lipid oxidation are emerging as important secondary messengers in tissue homeostasis. However, their regulation and mechanism of action remain poorly studied invivo during normal development. Here, we reveal that the fine regulation of hydrogen peroxide (H2O2) levels by its scavenger Catalase to mediate the switch from proliferation to differentiation in retinal progenitor cells (RPCs) is crucial. We identify 9-hydroxystearic acid (9-HSA), an endogenous downstream lipid peroxidation product, as a mediator of this effect in the zebrafish retina. We show that the 9-HSA proliferative effect is due to the activation of Notch and Wnt pathways through the inhibition of the histone deacetylase 1. We show that the local and temporal manipulation of H2O2 levels in RPCs is sufficient to trigger their premature differentiation. We finally propose a mechanism that links H2O2 homeostasis and neuronal differentiation via the modulation of lipid peroxidation.

Neuroprotective Effects of the Amylin Analog, Pramlintide, on Alzheimer's Disease Are Associated with Oxidative Stress Regulation Mechanisms.

Administration of the recombinant analog of the pancreatic amyloid amylin, Pramlintide, has shown therapeutic benefits in aging and Alzheimer's disease (AD) models, both on cognition and amyloid-β (Aβ) pathology. However, the neuroprotective mechanisms underlying the benefits of Pramlintide remain unclear. Given the early and critical role of oxidative stress in AD pathogenesis and the known reactive oxygen species (ROS) modulating function of amyloids, we sought to determine whether Pramlintide's neuroprotective effects involve regulation of oxidative stress mechanisms. To address this, we treated APP/PS1 transgenic mice with Pramlintide for 3 months, starting at 5.5 months prior to widespread AD pathology onset, and measured cognition (Morris Water Maze), AD pathology, and oxidative stress-related markers and enzymes in vivo. In vitro, we determined the ability of Pramlintide to modulate H2O2-induced oxidative stress levels. Our data show that Pramlintide improved cognitive function, altered amyloid-processing enzymes, reduced plaque burden in the hippocampus, and regulated endogenous antioxidant enzymes (MnSOD and GPx1) and the stress marker HO-1 in a location specific manner. In vitro, Pramlintide treatment in neuronal models reduced H2O2-induced endogenous ROS production and lipid peroxidation in a dose-dependent manner. Together, these results indicate that Pramlintide's benefits on cognitive function and pathology may involve antioxidant-like properties of this compound.