Marker Of Protein Oxidation Research Articles

Oxidative damage and mitochondrial functionality in hearts from KO UCP3 mice housed at thermoneutrality.

The antioxidant role of mitochondrial uncoupling protein 3 (UCP3) is controversial. This work aimed to investigate the effects of UCP3 on the heart of mice housed at thermoneutral temperature, an experimental condition that avoids the effects of thermoregulation on mitochondrial activity and redox homeostasis, preventing the alterations related to these processes from confusing the results caused by the lack of UCP3. WT and KO UCP3 mice were acclimatized at 30°C for 4weeks and hearts were used to evaluate metabolic capacity and redox state. Tissue and mitochondrial respiration, the activities of the mitochondrial complexes, and the protein expression of mitochondrial complexes markers furnished information on mitochondrial functionality. The levels of lipid and protein oxidative damage markers, the activity of antioxidant enzymes, the reactive oxygen species levels, and the susceptibility to in vitro Fe-ascorbate-induced oxidative stress furnished information on redox state. UCP3 ablation reduced tissue and mitochondrial respiratory capacities, not affecting the mitochondrial content. In KO UCP3 mice, the mitochondrial complexes activities were lower than in WT without changes in their content. These effects were accompanied by an increase in the level of oxidative stress markers, ROS content, and in vitro susceptibility to oxidative stress, notwithstanding that the activities of antioxidant enzymes were not affected by UCP3 ablation. Such modifications are also associated with enhanced activation/phosphorylation of EIF2α, a marker of integrated stress response and endoplasmic reticulum stress (GRP778 BIP). The lack of UCP3 makes the heart more prone to oxidative insult by reducing oxygen consumption and increasing ROS. Our results demonstrate that UCP3 helps the cell to preserve mitochondrial function by mitigating oxidative stress.

Integrative Biomarker Indices in a Benthic Indicator Species Modiolus Modiolus (L.) Under a Simulated Oil Spill

The horse mussel Modiolus modiolus (L.) is a sentinel bivalve species used in monitoring programs to assess potential biological exposure to anthropogenic contaminants, including oil hydrocarbons, in benthic environments. In an aquarium based experiment, these mussels were exposed to a simulated oil spill where crude oil at realistic concentrations (from low to high) interacted with an inert environment (seawater, gravel surface, etc.) and the biota. Using a combination of endpoints that included tissue contaminant load, protease activity, antioxidant enzyme activity, and low-molecular antioxidant profiles, we characterized M. modiolus responses to this simulated crude oil spill. Significant differences were observed in tissue protein reserves, protease activity, and oxidative stress markers including glutathione content and glutathione-S-transferase activity in the hepatopancreas and gill tissues of the bivalves treated with oil. Total concentrations of oil-derived polycyclic aromatic hydrocarbons in M. modiolus tissues were generally highest under the highest ambient oil concentration with much lower levels at the lowest. This general pattern does match the activity of protein quality control proteases but not the antioxidant enzyme activity profiles. Glutathione-S-transferase activity in bivalves showed decreased activity under the high compared to the lower oil load and oil-free animals, while glutathione content and calpain activity were positively correlated with oil uptake. The data suggest that these benthic organisms were exhibiting biological responses to the oil polycyclic aromatic hydrocarbons and support the bioindicator value of the species.
 Keywords: M. modiolus, oil spill, PAHs, integrative biomarker approach, protein quality control, GSH, GST

Biochemical Evaluation of Carbonic Anhydrase and Some Antioxidant Markers in Patients with Diabetes Complications.

Diabetes is a category of metabolic illnesses defined by a persistently high blood sugar level. This complication is caused by either the pancreas failing to create enough insulin or the body's cells failing to respond correctly to the insulin produced. Diabetes, if left untreated, can lead to a slew of health issues. Diabetic ketoacidosis, hyperosmolar hyperglycemia, and mortality are all examples of acute complications. There are numerous serious long-term consequences, including chronic renal disease, foot ulcers, as well as nerve and eye damage. This study aimed to extract carbonic anhydrase (CA) from human red blood cells and estimate the activity and specific activity of the enzyme and some biochemical parameters, including total protein, albumin, globulin, free amino acids, free amino acids/total protein (TP), thiol, thiol/TP, as well as carbonyl and carbonyl/TP levels in patients with diabetes complications, compared to the healthy subjects; moreover, it was attempted to investigate the correlation among the aforementioned variables. This study included 60 blood samples obtained from patients with diabetes complications and 40 healthy individuals as control. The results revealed a significant (P≤0.05) decrease in the TP levels, while the CA activity and specific activity were significantly (P≤0.05) increased. Moreover, there was a non-significant (P≥0.05) increase in the free amino levels; however, a significant (P≤0.05) increase was observed in albumin, free amino/TP, thiol, thiol/TP, as well as carbonyl and carbonyl/TP levels. On the other hand, a significant (P≤0.05) decrease was found in the levels of globulin and albumin/globulin ratio (AGR) in the patients, compared to the healthy subjects. The results also indicated a significant (P≤0.05) difference in all cases of diabetes mellitus (DM) complications for the measured parameters, except for the TP in DM nephropathy, albumin in cardiovascular disease, free amino in neuropathy and cardiovascular disease, and free amino/TP in retinopathy that showed a clear non-significant (P≥0.05) difference in the patients' groups, compared to the healthy subjects. The results of correlation indicated a significant (P≤0.05) positive correlation among free amino/TP, free amino/carbonyl, globulin/TP, and AGR/albumin. However, a significant negative correlation was noted between globulin/albumin and AGR/globulin. The results revealed that the protein oxidation markers and CA as antioxidant markers may play a role in monitoring diabetes complications.

DNA, protein and lipid oxidative damage in tissues of spontaneously hypertensive versus normotensive rats.

Oxidative damage to protein and lipid macromolecules in target organs in hypertension has been recognized as a major factor contributing to cardiovascular, cerebrovascular, and renal diseases. Data on protein and lipid oxidative damage in spontaneously hypertensive rats are numerous, but there is no information on DNA damage in tissues measured by comet assay. The aim of this study was to determine the baseline damage to DNA, protein, and lipid macromolecules in different organs of spontaneously hypertensive rats. Markers of lipid peroxidation, protein oxidation, and DNA damage were measured in blood, heart, kidney, and liver of 24-week-old spontaneously hypertensive rats. Plasma prooxidant and antioxidant status were determined as well. Age-matched normotensive Wistar rats were used as control. A rise in markers of lipid peroxidation and protein oxidation, malondialdehyde, and advanced oxidation protein products, was detected in all tissues of spontaneously hypertensive rats, with particularly high values in the liver. DNA damage, measured by the comet assay, was significantly higher in all the studied tissues of spontaneously hypertensive rats compared to normotensive control, with more severe damage in the cardiac and renal cells. Significant depletion of the plasma antioxidant barrier in spontaneously hypertensive rats was also observed. This study showed increased damage to all macromolecules in all studied samples of spontaneously hypertensive rats in comparison with control Wistar rats.

Effect of combined G6PD deficiency and diabetes on protein oxidation and lipid peroxidation

BackgroundOxidative Stress, an imbalance in the pro-oxidant/antioxidant homeostasis, occurs in many physiological and non-physiological processes and several human diseases, including diabetes mellitus (DM) and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Since the incidence of G6PD deficiency in Jordan and many parts of the world is high, this study aimed to measure the effect of G6PD deficiency on the oxidative markers and the antioxidant glutathione (GSH) in diabetic and non-diabetic individuals.MethodsWhole blood G6PD deficiency was screened by the fluorescent spot method, and erythrocyte G6PD activity was determined using a quantitative assay. Since protein carbonyl (PC) and malondialdehyde (MDA) are the most widely measured markers for protein and lipid oxidation, respectively, plasma PC and MDA, in addition to blood GSH were determined by spectrophotometric assays, as biomarkers of oxidative stress.ResultsThe incidence of G6PD deficiency among the diabetic subjects was 15%. PC level in patients with diabetes and in G6PD-deficient subjects was 5.5 to 6-fold higher than in non-diabetic subjects with sufficient G6PD levels (p<0.001). This fold increase was doubled in diabetic patients with G6PD deficiency (p<0.001). Furthermore, the MDA level was significantly increased by 28-41% in G6PD-deficient, diabetics with sufficient G6PD, and diabetics with G6PD deficiency compared to MDA level in non-diabetic with sufficient G6PD. On the other hand, GSH was significantly reduced to half in G6PD-deficient subjects and in diabetics with G6PD-deficiency.ConclusionsThe results showed that diabetes and G6PD deficiency increased protein oxidation and lipid peroxidation. However, the combination of both disorders has an additive effect only on protein oxidation. On the other hand, GSH level is only reduced in G6PD deficiency. In addition, diabetes and G6PD deficiency appear to be genetically linked since the incidence of G6PD deficiency among people with diabetes is more than the general population.

Lycopene Regulates Dietary Dityrosine-Induced Mitochondrial-Lipid Homeostasis by Increasing Mitochondrial Complex Activity.

Dityrosine (DT), a marker of protein oxidation, is widely found in many high-protein foods. Dietary intake of DT induces myocardial oxidative stress injury and impairs energy metabolism. Lycopene is a common dietary supplement with antioxidant and mitochondrial-lipid homeostasis modulating abilities. This study aimed to examine the effects of lycopene on DT-induced disturbances in myocardial function and energy metabolism. Four-week-old C57BL/6J mice received intragastric administration of either tyrosine (420µg kg-1 BW), DT (420µg kg-1 BW), or lycopene at high (10mg kg-1 BW) and low (5mg kg-1 BW) doses for 35 days. Lycopene administration effectively reduced oxidative stress, cardiac fatty acid accumulation, and cardiac hypertrophy and improved mitochondrial performance in DT-induced mice. In vitro experiments in H9c2 cells showed that DT directly inhibited the activity of the respiratory chain complex, whereas oxidative phosphorylation and β-oxidation gene expression is upregulated. Lycopene enhanced the activity of the complexes and inhibited ROS production caused by compensatory regulation. Lycopene improves DT-mediated myocardial energy homeostasis disorder by promoting the activity of respiratory chain complexes I and IV and alleviates the accumulation of cardiac fatty acids and myocardial hypertrophy.

Antioxidant and prooxidant activities of tea polyphenols in oil-in-water emulsions depend on the level used and the location of proteins

We studied the impacts of protein location (interface or aqueous phase) on the antioxidant and prooxidant activities of tea polyphenols (TP) in model oil-in-water emulsions (pH 7) at a low (0.01% w/v) or high (0.04 % w/v) concentration. TP at 0.01% reduced the levels of both lipid and protein oxidation markers in emulsions, independent of the protein location. However, TP were more potent when proteins were located at the interface. At 0.04%, TP were only weakly antioxidant towards lipids but were prooxidant towards proteins in emulsions with proteins at the interface, whereas they were still somewhat antioxidant for aqueous phase proteins. These results indicate that TP may act as either antioxidants or prooxidants depending on their concentration and also on the location of the proteins in emulsions. The level of TP should be optimized for emulsion-based foods or beverages to achieve optimum antioxidant activity.

Both spontaneous vaginal delivery and elective caesarean section influence neonatal redox status in dogs

The influence of birth mode on redox balance is unknown in dogs. We aimed to compare antioxidant activity and oxidative stress of puppies born by elective c-section or vaginal delivery. Twenty-one bitches were elected for spontaneous vaginal delivery (n=10) and elective cesarean section (n=11) groups, considering only the first and last puppies born. Neonatal oxygen saturation, lactatemia, superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase (GPx) and total antioxidant capacity; marker of oxidative stress (thiobarbituric acid reactive substances [TBARS]) and protein oxidation were analysed at birth, 1h, 12h, 24h and 72 h. C-section newborns had lower oximetry at birth and 1h. Lactatemia was higher in the last vaginal delivery puppies and higher at birth, and 1h. Regardless of birth mode, last puppies had hyperlactatemia at birth. Vaginal delivery puppies had higher TBARS, while protein oxidation was higher in c-section, mostly in last puppies. C-section puppies had higher GPx activity, whereas GSH concentration decreased overtime. Surgically born puppies had lowering SOD between 12h and 72h. Vaginal delivery enhances lipid peroxidation, counteracted mostly by GPx. Conversely, c-section triggers protein oxidation, acutely scavenged by SOD and GSH, thus puppies are less exposed to the negative consequences of oxidative stress.

Pharmacologic Ascorbate Enhances the Therapeutic Index of ATM-Inhibitor Based Chemoradiation for Colorectal Cancer

Ataxia telangiectasia mutated protein (ATM) is one of the key sensors of DNA damage and specific inhibitors of ATM are potent radiosensitizers. However, their clinical utility with radiation (RT) is limited because they lack tissue specificity and increase normal tissue injury. Pharmacologic (high dose) ascorbate (P-AscH-) selectively increases oxidative stress in tumors while functioning as a donor antioxidant and reducing RT damage in normal tissues. We hypothesized that P-AscH- could enhance the therapeutic index of ATM-inhibitor based chemoradiation (CRT) for colorectal cancer (CRC) by simultaneously enhancing efficacy and reducing RT bowel injury.Human HCT116, SW480, and HT29 and murine CT26 and MC38 CRC models were used. Clonogenic survival was assessed following single-fraction RT (2-8 Gy) +/- P-AscH- (5 pM/cell) +/- veliparib (PARP), VE821 (ATR), or KU60019 (ATM). Catalase expression was induced using HCT116 cells expressing a doxycycline inducible catalase transgene. DNA double strand breaks (DSBs) were quantified using neutral comet assays 0-24 hours post RT. Cell cycle phases were assessed using flow cytometry. ATM and pATM localization were assessed using IF. Jejunal toxicity was assessed using IHC in fixed tissues following single fraction (10 Gy) whole abdominal RT in c57bl/6 mice. Tumor growth delay was assessed following RT (5 Gy x 3) +/- drug treatment in unilateral flank tumors.Veliparib, VE821, and KU60019 were potent radiosensitizers in HCT116, SW480, HT29, MC38, and CT26 CRC tumor models and P-AscH- further reduced clonogenic survival with DRIs in all lines except for HT29. In contrast, P-AscH- enhanced survival of cultured HUVEC and FHs-74 cells exposed to RT. Enhanced cell kill with P-AscH- is H202 mediated as it is completely attenuated by inducible catalase expression. P-AscH- significantly increased the number of DNA DSBs in tumors after RT in vitro. Despite the increase in DNA DSBs, P-AscH-significantly decreased nuclear localization of activated pATM after RT and significantly decreased the fraction of cells in G2/M phases of the cell cycle. In vivo, RT + P-AscH- + KU60019 induced more tumor growth delay/clearance than all other combinations in unilateral MC38 or HCT116 flank tumors. Finally, P-AscH- significantly reduced loss of jejunal crypt cell density, epithelial architecture, and markers of lipid and protein oxidation following whole abdominal RT.P-AscH- selectively enhances the efficacy of ATM-based CRT in CRC tumor models while simultaneously decreasing RT-mediated small bowel toxicity. In tumors, P-AscH- enhances DNA DSBs by stimulating an H202 flux and prevents activation of DNA repair pathways and cell cycle checkpoints by inhibiting RT-induced activation of ATM. Selective radioprotectors like P-AscH- could facilitate the clinical translation ATM inhibitors as radiosensitizers.

Protein carbonylation in food and nutrition: a concise update

Protein oxidation is a topic of indisputable scientific interest given the impact of oxidized proteins on food quality and safety. Carbonylation is regarded as one of the most notable post-translational modifications in proteins and yet, this reaction and its consequences are poorly understood. From a mechanistic perspective, primary protein carbonyls (i.e. α-aminoadipic and γ-glutamic semialdehydes) have been linked to radical-mediated oxidative stress, but recent studies emphasize the role alternative carbonylation pathways linked to the Maillard reaction. Secondary protein carbonyls are introduced in proteins via covalent linkage of lipid carbonyls (i.e. protein-bound malondialdehyde). The high reactivity of protein carbonyls in foods and other biological systems indicates the intricate chemistry of these species and urges further research to provide insight into these molecular mechanisms and pathways. In particular, protein carbonyls are involved in the formation of aberrant and dysfunctional protein aggregates, undergo further oxidation to yield carboxylic acids of biological relevance and establish interactions with other biomolecules such as oxidizing lipids and phytochemicals. From a methodological perspective, the routine dinitrophenylhydrazine (DNPH) method is criticized not only for the lack of accuracy and consistency but also authors typically perform a poor interpretation of DNPH results, which leads to misleading conclusions. From a practical perspective, the biological relevance of protein carbonyls in the field of food science and nutrition is still a topic of debate. Though the implication of carbonylation on impaired protein functionality and poor protein digestibility is generally recognized, the underlying mechanism of such connections requires further clarification. From a medical perspective, protein carbonyls are highlighted as markers of protein oxidation, oxidative stress and disease. Yet, the specific role of specific protein carbonyls in the onset of particular biological impairments needs further investigations. Recent studies indicates that regardless of the origin (in vivo or dietary) protein carbonyls may act as signalling molecules which activate not only the endogenous antioxidant defences but also implicate the immune system. The present paper concisely reviews the most recent advances in this topic to identify, when applicable, potential fields of interest for future studies.

First-degree relatives of type-2 diabetic patients and dysglycaemic patients have impaired endothelial function due to decreased bioavailability of nitric oxide

Abstract Background/Introduction Oxidative stress plays an important role in the pathogenesis of type 2 diabetes. Purpose We investigated oxidative stress in first-degree relatives of type-2 diabetes patients (FDR) since they are more prone to develop type-2 diabetes, at baseline and during postprandial hyperglycemia in comparison with dysglycaemic or normoglycaemic subjects. We evaluated these results in relation to vascular function. Methods We studied 40 FDR with normal oral glucose test (OGTT), 40 subjects with abnormal OGTT (dysglycaemic) and 20 subjects with normal OGTT without parental history of diabetes (normoglycaemic) with similar demographical and clinical characteristics. Glucose and insulin levels, pulse wave velocity (PWV-Complior, ALAM), central systolic blood pressure (cSBP) and augmentation index (AI) were measured at baseline, 30, 60, 90 and 120min during OGTT. Perfused boundary region (PBR-Microscan, Glycocheck) of the sublingual arterial microvessels (high PBR values represent reduced glycocalyx thickness), plasma concentrations of malondialdehyde (MDA) and protein carbonyls (PCs) as markers of lipid peroxidation and protein oxidation, respectively, and nitrite/nitrates levels as markers for NO biosynthesis, were assessed at baseline and at 120min of OGTT. Insulin sensitivity was evaluated using Matsuda and insulin sensitivity index (ISI). Results FDR and dysglycaemics patients had higher fasting insulin, reduced ISI, Matsuda index as well as increased PBR (2.5±0.5 vs. 2.5±0.6 vs. 2.4±0.3μm), increased PWV (8.9±1.1 vs. 10.3±2.4 vs. 8.0±1.5m/s), AI, MDA, PCs and nitrite/nitrate than normoglycaemic subjects (p&amp;lt;0.05 for all comparisons). ISI was negatively related with PBR and MDA (r=−0.35 and r=−0.34, p&amp;lt;0.05) at baseline in FDR and dysglycaemics. During OGTT, AI was similarly reduced in both normoglycaemics and FDR whereas AI was significantly increased in dysglycaemics at 120min (p&amp;lt;0.05). PBR was increased by 7.5% and 4% at 120min in dysglycaemics and FDR, respectively, while remained unchanged in normoglycaemics (p&amp;lt;0.05). In dysglycaemics and FDR, nitrite/nitrate levels were significantly decreased at 120min (52.85±8.22 vs. 36.31±4.36 and 48.23±7.20 vs. 34.70±4.71nmol/ml, respectively, p&amp;lt;0.05) while they were remained unchanged in normoglycaemics likely leading to a greater decrease of MDA (−83% vs. −40% vs. −48%) and of PCs (−28% vs. −7% vs. −16%) in normoglycaemics compared with dysglycaemics and FDR (p&amp;lt;0.05). Conclusions Insulin resistance determines acute endothelial responses during postprandial hyperglycemia leading to reduced NO production and thus facilitating augmentation of oxidative stress during postprandial hyperglycemia in dysglycaemic patients and FDR of diabetic patients. Funding Acknowledgement Type of funding sources: None.

Selective and sensitive UHPLC-ESI-Orbitrap MS method to quantify protein oxidation markers

A targeted UHPLC-MS/MS isotopic dilution method has been developed for the simultaneous quantification of 18 different free and protein-bound aromatic amino acid oxidation products in food and biological matrices. All analytes, including critical isomeric pairs of Tyr, o-Tyr, m-Tyr, and dioxyindolylalanine diastereomers were chromatographically resolved to obtain high selectivity, without the need for derivatizing or ion pairing agents. The results of method validation showed adequate retention time reproducibility [0.1–0.6% coefficient of variation (CV) for over 224 injections], accuracy (within ±1–20% of the nominal concentration), and precision (1–17% CV) for all target analytes. The lower limit of quantification was calculated in different matrices using both the Hubaux-Vos approach and accuracy and precision data showing values in the range of 0.2–15 ng/mL. Use of stable isotope-labelled internal standards compensated errors due to matrix effects and artefactual degradation of analytes. Both acid and enzymatic hydrolyses were tested to obtain the best possible results for the quantification of protein oxidation products, demonstrating the stability of target analytes under hydrolytic conditions. The method was successfully applied to quantify target analytes in serum, tissue, milk, infant formula, pork liver pâté, chicken meat and fish. The method was also applied to assess the role of Fenton's reagent in oxidizing Trp, Phe and Tyr residues in different proteins, with results showing o-Tyr, dioxyindolylalanine diastereomers, kynurenine, dityrosine being the main oxidation products. The Fenton chemistry favored the formation of o-Tyr over m-Tyr from Phe with 2–36 folds higher yields. 3-Nitrotyrosine, a marker of protein nitration, was also detected in samples treated with Fenton's reagent.

Effects of commonly used medicinal herbs in Jordan on erythrocyte oxidative stress markers

Context: Much attention has been given recently to the antioxidant capacity of natural products, with particular interest on those that are frequently consumed by people. Aims: To evaluate the commonly used and frequently consumed edible herbs in Jordan to compare their in vitro and in vivo antioxidant properties. Methods: The in vitro antioxidant properties were tested by pre-incubating washed human erythrocytes with a given herb extract and then exposing these erythrocytes to H2O2 to induce oxidative stress, and then measuring erythrocyte malondialdehyde (MDA) as a marker for lipid peroxidation, protein carbonyl (PC) as a marker for protein oxidation, reduced glutathione (GSH) as a marker for cellular antioxidant status and the percentage of hemolysis as an indicator for the anti-hemolytic activity of the herb. The in vivo antioxidant properties were tested by giving orally aqueous extracts of the herbs tested in vitro to healthy individuals on daily bases for five days, with two blood samples being collected from each individual to measure the above-mentioned markers. Results: Pre-incubation of human erythrocytes in vitro with methanolic extracts of Zingiber officinale, Rosmarinus officinalis, Salvia triloba, Verbena triphylla, Nigella sativa and Origanum syriacum significantly improved erythrocyte MDA, PC and oxidant hemolysis. Oral consumption by healthy individuals of aqueous extracts of the same herbs for 5 days significantly improved erythrocyte MDA, GSH, and superoxide dismutase (SOD) at the sixth day of administration. Conclusions: These results indicate that aqueous extracts of medicinal herbs can be absorbed well and appear in body tissues inflecting in vivo antioxidant properties similar to their in vitro properties.

Pharmacologic ascorbate enhances the therapeutic index of ATM-inhibitor based chemoradiation for colorectal cancer.

3609 Background: Ataxia telangectasia mutated protein (ATM) is one of the key sensors of DNA damage and specific inhibitors of ATM are potent radiosensitizers. However, their clinical utility with radiation (RT) is limited because they lack tissue specificity and increase normal tissue injury. Pharmacologic (high dose) ascorbate (P-AscH-) selectively increases oxidative stress in tumors while functioning as a donor antioxidant and reducing RT damage in normal tissues. We hypothesized that P-AscH- could enhance the therapeutic index of ATM-inhibitor based chemoradiation (CRT) for colorectal cancer (CRC) by simultaneously enhancing efficacy and reducing RT bowel injury. Methods: Human HCT116, SW480, and HT29 and murine CT26 and MC38 CRC models were used. Clonogenic survival was assessed following single-fraction RT (2-8 Gy) +/- P-AscH- (5 pM/cell) +/- veliparib (PARP), VE821 (ATR), or KU60019 (ATM). Catalase expression was induced using HCT116 cells expressing a doxycycline inducible catalase transgene. DNA double strand breaks (DSBs) were quantified using neutral comet assays 0-24 hours post RT. Cell cycle phases were assessed using flow cytometry. ATM and pATM localization were assessed using IF. Jejunal toxicity was assessed using IHC in fixed tissues following single fraction (10 Gy) whole abdominal RT in c57blj/6 mice. Tumor growth delay was assessed following RT (5 Gy x 3) +/- drug treatment in unilateral flank tumors. Results: Veliparib, VE821, and KU60019 were potent radiosensitizers in HCT116, SW480, HT29, MC38, and CT26 CRC tumor models and P-AscH- further reduced clonogenic survival with DRIs in all lines except for HT29. In contrast, P-AscH- enhanced survival of cultured HUVEC and FHs-74 cells exposed to RT. Enhanced cell kill with P-AscH- is H202 mediated as it is completely attenuated by inducible catalase expression. P-AscH- significantly increased the number of DNA DSBs in tumors after RT in vitro. Despite the increase in DNA DSBs, P-AscH-significantly decreased nuclear localization of activated P-ATM after RT and significantly decreased the fraction of cells in G2/M phases of the cell cycle. In vivo, RT + P-AscH- + KU60019 induced more tumor growth delay/clearance than all other combinations in unilateral MC38 or HCT116 flank tumors. Finally, P-AscH- significantly reduced loss of jejunal crypt cell density, epithelial architecture, and markers of lipid and protein oxidation following whole abdominal RT. Conclusions: P-AscH- selectively enhances the efficacy of ATM-based CRT in CRC tumor models while simultaneously decreasing RT-mediated small bowel toxicity. In tumors, P-AscH- enhances DNA DSBs by stimulating an H202 flux and prevents activation of DNA repair pathways and cell cycle checkpoints by inhibiting RT-induced activation of ATM. Selective radioprotectors like P-AscH- could facilitate the clinical translation ATM inhibitors as radiosensitizers.

The effect of hypothermia in a rat testicular torsion/detorsion model

Testicular torsion is an emergent condition. The protective effect of medical hypothermia in ischemia/reperfusion injury is well defined. To evaluate the late results of hypothermia through a rat testicular torsion/detorsion model compatible with human testicular torsion. Rats were divided into 5 groups (n=7): (1)Sham (S) group, (2)T/D group: right testis was torted for 1-h, (3)T/D+H30 group: hypothermia at 4°C was applied for 30min before detorsion, (4)T/D+H90 group: hypothermia at 4°C was applied for a total of 90min (30min before and 1-h after detorsion), (5)H group: hypothermia at 4°C was applied to right testis for 90min. Testicular diameters at preoperative period and 8th postoperative week were measured. Biochemically, MPO, NO, 3-NT and 4-HNE in testicular tissue and serum levels of NO, PGF 2α, 3-NT, 8-OHdG and 4-HNE were studied. Histopathologic examination and TUNEL assay were also performed. Biochemical and macroscopical parameters of both T/D+H30 and T/D+H90 groups were statistically different from group T/D with respect to protective effects of hypothermia. Johnsen's score was also statistically different in group T/D+H90. Hypothermia can easily be applied with ice bags both in perioperative period. This is the first study evaluating the effect of hypothermia applied postoperatively. Tissue level of protein oxidation marker (3-NT) and serum levels of DNA damage (8-OHdG), lipid peroxidation (4-HNE), protein oxidation (3-NT) and oxidative stress (PGF-2α) markers were measured for the first time. Hypothermia has been shown to be macroscopically, biochemically and histopathologically beneficial in the long-term experimental testicular torsion model.

Protective effects of dietary carnosine during in-vitro digestion of pork differing in fat content and cooking conditions.

Muscle carnosine represents an important health advantage of meat. Ground pork samples with intrinsic or added carnosine; fat content; and cooked under low or high intensity as a 2×2×2 factorial were digested in-vitro. Changes in free carnosine and in markers of lipid (hexanal, 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and protein (protein-carbonyls, thiols) oxidation, and of advanced glycation end-products (AGEs) Nε -(carboxymethyl)lysine (CML) were determined in the saliva, gastric, and duodenal digests. During digestion, the different markers overall indicated increased oxidation and decreased free carnosine. Increasing pork carnosine level significantly reduced protein carbonyls, loss of thiols, and 4-HNE during in-vitro gastric digestion, irrespective of fat and cooking level of the meat. Increased carnosine also significantly reduced hexanal, MDA and CML up to the duodenum phase in moderately cooked lean pork. Besides substantiating the formation of AGEs during digestion, these results show a potentially important role of dietary carnosine occurring in the gastrointestinal tract. PRACTICAL APPLICATIONS: The ailments epidemiologically associated with red meat consumption could be counteracted by ingesting carnosine into meat. The health advantages of dietary carnosine, however, have never been demonstrated during digestion, a unique and complex oxidative environment compounded by the composition and cooking of the meat. The results obtained substantiated that AGEs formation occurred in-vitro in the GIT. They also showed that increased carnosine had an immediate health beneficial role during pork digestion in reducing the formation of different harmful molecules, including AGEs, modulated by the composition and cooking of the meat. However, in exerting this protective role in the GIT, the remaining free level of carnosine, gradually decreased during digestion. Carnosine, as an important meat compositional factor may, depending on the fat content and cooking conditions, change the image of meat from representing a health risk to a health benefit. Carnosine level may also explain discrepancies observed in the literature.

Roux-en-Y Gastric Bypass and Sleeve Gastrectomy Differently Affect Oxidative Damage Markers and their Correlations with Body Parameters.

Bariatric surgery improves oxidative damage, but little is known about the differences between Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG). This study compared changes in lipid and protein oxidative damage markers and their correlations with body parameters of patients before and after RYGB or SG. Body mass index (BMI), bioimpedance parameters, and biochemical parameters including lipid and protein oxidative damage markers were evaluated before and 6months after surgery. Data were analyzed by t test or Mann-Whitney rank sum test and Spearman's correlation coefficient between oxidative damage and other parameters. Twenty-five patients were submitted to RYGB and 14 to SG. There was a significant decrease of BMI, fat mass, fat-free mass, phase angle, serum total protein, transthyretin, and C-reactive protein in both groups (p< 0.05). Serum thiobarbituric acid reactive substances (TBARS), advanced oxidation protein products (AOPP), and serum lipids (p < 0.05) were significantly decreased in the RYGB group. TBARS levels were significantly correlated with serum total cholesterol (r = 0.468), LDL (r = 0.439), BMI (r = 0.424), and fat mass (r = 0.40) (p< 0.05). In the SG group, AOPP levels were significantly correlated with serum C-reactive protein (baseline: r = 0.53, 6months: r = 0.64) (p < 0.05). Alterations in these levels were negatively correlated with changes in BIA parameters [resistance (r = -0.574), reactance (r = -0.736), and phase angle (r = 0.549)] (p < 0.05). RYGB seems to be better in attenuating oxidative damage after 6months. The BMI reduction in the RYGB group suggests a concomitant decrease of lipid oxidative damage. In the SG group, changes in BIA parameters were inversely correlated with protein oxidative damage.

A Protective Strategy to Counteract the Oxidative Stress Induced by Simulated Microgravity on H9C2 Cardiomyocytes.

Microgravity affects human cardiovascular function inducing heart rhythm disturbances and even cardiac atrophy. The mechanisms triggered by microgravity and the search for protection strategies are difficult to be investigated in vivo. This study is aimed at investigating the effects induced by simulated microgravity on a cardiomyocyte-like phenotype. The Random Positioning Machine (RPM), set in a CO2 incubator, was used to simulate microgravity, and H9C2 cell line was used as the cardiomyocyte-like model. H9C2 cells were exposed to simulated microgravity up to 96 h, showing a slower cell proliferation rate and lower metabolic activity in comparison to cell grown at earth gravity. In exposed cells, these effects were accompanied by increased levels of intracellular reactive oxygen species (ROS), cytosolic Ca2+, and mitochondrial superoxide anion. Protein carbonyls, markers of protein oxidation, were significantly increased after the first 48 h of exposition in the RPM. In these conditions, the presence of an antioxidant, the N-acetylcysteine (NAC), counteracted the effects induced by the simulated microgravity. In conclusion, these data suggest that simulated microgravity triggers a concomitant increase of intracellular ROS and Ca2+ levels and affects cell metabolic activity which in turn could be responsible for the slower proliferative rate. Nevertheless, the very low number of detectable dead cells and, more interestingly, the protective effect of NA, demonstrate that simulated microgravity does not have “an irreversible toxic effect” but, affecting the oxidative balance, results in a transient slowdown of proliferation.

Lipid and Protein Oxidation Marker Compounds in Horse Meat Determined by MIR Spectroscopy.

This work broadens the study of lipid and protein oxidation marker compounds in foal meat, employing the technology of Attenuated Total Reflectance-Fourier Transform Mid-Infrared Spectroscopy (ATR-FT/MIR, shortened in the following as MIR). As a main objective, marker compounds from 23 foals were extracted and their absorbance spectra were measured to establish prediction models (calibration and validation) between them and classical quantification analysis of the compounds. Another objective was to ascertain whether a previous extraction of the marker compounds before executing their MIR analysis is preferable compared to direct MIR measurements on the raw meat samples. In this context, marker compound results (TBARS between 0.4387 and 2.1040, and carbonyls between 4.07 and 4.68) showed more consistent predictive models than the ones achieved using quantitative analysis of the spectra obtained from the raw meat. Lipid oxidation compounds predictive models obtained in this work offered an R2cv of 63.18% and protein oxidation R2cv obtained in this project showed a value of 54.24%. Thus, MIR technology arises as a promising tool to identify and quantify products derived from lipid and protein oxidation in fresh foal meat.

Alterations in oxidative metabolism in liver of female rats: Effects of long-term vitamin A deficiency

Background: The latest estimate by 5 UN agencies is that 821 million people globally are undernourished, which puts them at risk of vitamin and other nutrient deficiencies. Vitamin A deficiency remains a widespread public health problem among women and children in the developing world the role of vitamin A and its active metabolites in pathways involved in antioxidant protection and in the inhibition of important pathways that promote oxidative stress. Objectives: Determine if vitamin A deficiency could influence oxidative metabolism in 6-month-old female wistar rats. Materials and Methods: Determine the concentration of carbonyl proteins, as a marker of protein oxidation; TBARS, as a lipoperoxidation marker; and nitrotyrosine as a marker of oxidative stress dependent on nitric oxide. Quantify the expression of CAT, SOD, eNOS and iNOS in the liver and wistar rats deficient in vitamin A for 6 months. Results: An increase in the concentration of carbonyl protein and nitrotyrosine in the liver tissue deficient in vitamin A is observed. The expression of SOD, eNOS and iNOS decreased in the group with a private diet of vitamin A. From the regression analysis a positive correlation was established between hepatic retinoic acid levels and gene expression of eNOS, iNOS and SOD. A positive correlation between serum retinoic acid levels and gene expression of eNOS and iNOS was also observed. Conclusions: It is possible to ratify the relationship between the development of stress and vitamin A levels; improving the understanding of hepatic metabolism and its response to the absence of this vitamin.