Oxidative Macromolecular Alterations in the Rat Central Nervous System in Response to Experimentally Co-Induced Chlorpyrifos and Cold Stress: A Comparative Assessment in Aging Rats

Reactive oxygen species are generated as a result of a number of physiological and pathological processes which can promote multiple forms of oxidative damage including protein oxidation, and thereby influence the function of a diverse array of cellular processes. In our previous study we have reported that co-exposure to chlorpyrifos and cold stress in aging rats markedly influence the toxic outcome as a result of oxidative stress. In the present study, key neurochemical/enzymes were measured in order to evaluate the macromolecular alterations in response to experimentally co-induced chlorpyrifos and cold stress (15 and 20°C) either concurrently or individually in vivo for 48 h in discrete regions of brain and spinal cord of different age group rats. CPF and cold stress exposure either individually or in combination substantially increased the activity/levels of protein carbonyls, AST, ALT and decreased protein thiols, DNA, RNA and total proteins in discrete regions of CNS. Overall, the effects of co-exposure were appreciably different from either of the exposures. However, synergistic-action of CPF and cold stress at 15°C showed higher dyshomeostasis in comparison with CPF and cold stress alone and together at 20°C indicating the extent of oxidative macromolecular damage in discrete regions of brain and spinal cord. Furthermore, the present study demonstrates that macromolecular oxidative damage is highly pronounced in neonates and juveniles than the young adults.     

Sub-lethal effect of synthetic pyrethroid pesticide on metabolic enzymes and protein profile of non-target Zebra fish,Danio rerio

Extensive application of pesticide in agricultural field affects the enzymatic activity of non-target animals, including fishes. In this study, the impact of sublethal concentration of fenvalerate on marker enzymes of freshwater Zebra fish was evaluated. Pesticide-induced stress can specifically affect non target fishes, through elevated level of reactive oxygen species which is responsible for biochemical, cell metabolism and physiological activities. The oxidative stress mediated by fenvalerate at sub lethal concentrations after 28 days of exposure of Zebra fish. Following 28 days of exposure of pesticide, catalase, superoxide dismutase, aspartate amino transferases, alanine amino transferase, alkaline phosphatase and acid phosphatase were assessed. Results revealed reduction of superoxide dismutase activity after 28 days of exposure in sub lethal concentration of fenvalerate in liver and gills. In liver, catalase activity was found to be less in fenvalerate exposed fish than control fish. In liver, increase of 75.75% aspartate amino transferase and 38% increase in alanine amino transferase in gills. SGPT activity was relatively higher than SGOT suggests more contribution of phyruvalate than oxaloacetate formation. Fenvalerate induced changes in acid phosphatase and alkaline phosphatase activity in the liver and gills of Zebra fish after four weeks of exposure. Fenvalerate induced expression of various stress proteins in gill, liver, followed by muscle. Some proteins lost its intensity due to fenvalerate toxicity. Result revealed that enzyme assays and SDS-PAGE analysis for protein subunits determination is relevant tool to monitor stress in freshwater ecosystem. The findings suggest that in monitoring fenvalerate toxicity programme, enzyme activities can be potent diagnostic tool for fenvalerate induced toxicity.     

Mitochondrial Dysfunction in Aging Rat Brain Regions upon Chlorpyrifos Toxicity and Cold Stress: An Interactive Study

Mitochondrial dysfunction and consequent energy depletion are the major causes of oxidative stress resulting to bring alterations in the ionic homeostasis causing loss of cellular integrity. Our previous studies have shown the age-associated interactive effects in rat central nervous system (CNS) upon co-exposure to chlorpyrifos (CPF) and cold stress leading to macromolecular oxidative damage. The present study elucidates a possible mechanism by which CPF and cold stress interaction cause(s) mitochondrial dysfunction in an age-related manner. In this study, the activity levels of Krebs cycle enzymes and electron transport chain (ETC) protein complexes were assessed in the isolated fraction of mitochondria. CPF and cold stress (15 and 20 °C) exposure either individually or in combination decreased the activity level of Krebs cycle enzymes and ETC protein complexes in discrete regions of rat CNS. The findings confirm that cold stress produces significant synergistic effect in CPF intoxicated aging rats. The synergism between CPF and cold stress at 15 °C caused a higher depletion of respiratory enzymes in comparison with CPF and cold stress alone and together at 20 °C indicating the extent of deleterious functional alterations in discrete regions of brain and spinal cord (SC) which may result in neurodegeneration and loss in neuronal metabolic control. Hence, co-exposure of CPF and cold stress is more dangerous than exposure of either alone. Among the discrete regions studied, the cerebellum and medulla oblongata appears to be the most susceptible regions when compared to cortex and SC. Furthermore, the study reveals a gradual decrease in sensitivity to CPF toxicity as the rat matures.     

Comparative proteomic and metabolomic analyses reveal mechanisms of improved cold stress tolerance in bermudagrass (Cynodon dactylon (L.) Pers.) by exogenous calcium

As an important second messenger, calcium is involved in plant cold stress response, including chilling (<20 °C) and freezing (<0 °C). In this study, exogenous application of calcium chloride (CaCl₂) improved both chilling and freezing stress tolerances, while ethylene glycol‐bis‐(β‐aminoethyl) ether‐N,N,N,N‐tetraacetic acid (EGTA) reversed CaCl₂ effects in bermudagrass (Cynodon dactylon (L.) Pers.). Physiological analyses showed that CaCl₂ treatment alleviated the reactive oxygen species (ROS) burst and cell damage triggered by chilling stress, via activating antioxidant enzymes, non‐enzymatic glutathione antioxidant pool, while EGTA treatment had the opposite effects. Additionally, comparative proteomic analysis identified 51 differentially expressed proteins that were enriched in redox, tricarboxylicacid cycle, glycolysis, photosynthesis, oxidative pentose phosphate pathway, and amino acid metabolisms. Consistently, 42 metabolites including amino acids, organic acids, sugars, and sugar alcohols were regulated by CaCl₂ treatment under control and cold stress conditions, further confirming the common modulation of CaCl₂ treatment in carbon metabolites and amino acid metabolism. Taken together, this study reported first evidence of the essential and protective roles of endogenous and exogenous calcium in bermudagrass response to cold stress, partially via activation of the antioxidants and modulation of several differentially expressed proteins and metabolic homeostasis in the process of cold acclimation.     

Effects of cold stress on juvenile Piaractus mesopotamicus and the mitigation by β-carotene

This study investigated the effects of cold stress on morphometrical and hematological biomarkers, energy metabolism, and oxidative stress in different tissues of P. mesopotamicus, and the protective role of β-carotene. Fish were fed with a control diet (CD) and the same diet supplemented with 105 mg/kg β-carotene (BD) for 60 days. After the feeding trial, fish fed CD or BD diets were exposed to control (24 °C) and low temperature (14 °C) for 24 h. Fish (CD and BD) exposed to thermal stress showed lower hepatosomatic index. The hemoglobin increased only in CD-fed fish exposed to 14 °C. Increased glycemia, plasmatic protein depletion, and decreased hepatic glycogen were observed in fish fed the CD, while only the lipid levels in liver were augmented in BD-fed fish exposed at 14 °C. Regarding the oxidative stress, increased antioxidant enzymes activity and lipid peroxidation were observed in CD-fed fish exposed to cold. The two-way ANOVA showed an interaction between dietary treatment and temperature for glucose and oxidative stress biomarkers, with the highest values recorded in 14 °C-exposed fish fed with the CD. Our study demonstrated that cold stress had the greatest impact on fish oxidative status, and β-carotene reduces harmful effects induced by cold in P. mesopotamicus.     

A proteomic analysis to identify cold acclimation associated proteins in wild wheat (Triticum urartu L.)

To gain a better understanding of cold acclimation process in wheat, we applied a 2-DE based proteomic approach to discover changes in proteome profile of a diploid wild wheat (Triticum urartu L.) during prolonged cold stress treatment. To this end, plants were grown in pots and the growing seedlings (4-leaf stage) were exposed to cold stress. After 4 weeks of cold acclimation (4–6 °C) and subsequent treatment for 12 h at ?2 °C, samples were collected from control and stressed plants and were subjected to proteome pattern analysis. Among approximately 450 reproducible protein spots displayed in each given 2-DE gels, 34 proteins changed significantly in abundance in response to cold stress. Among them, 25 and 9 proteins were up and down-regulated under stress condition, respectively. Analysis by matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry coupled with non-redundant protein database search allowed the identification of 20 cold-induced proteins. Integrated proteomic and database survey resulted in identification of several cold stress related proteins such as pathogenesis related protein, cold regulated protein, cold-responsive LEA/RAB-related COR protein, oxygen-evolving enhancer protein and oxalate oxidase. The presumed functions of the identified proteins were mostly related to cold acclimation, oxidative stress and photosynthesis. The possible implications of differentially accumulated proteins in acquiring systemic tolerance to freezing stress following exposure to prolonged low temperature will be discussed.     

Activity of Stress-related Antioxidative Enzymes in the Invasive Plant Crofton Weed （Eupatorium adenophorum）

Crofton weed is an invasive weed in southwestern China. The activities of several antioxidative enzymes involved in plant protection against oxidative stress were assayed to determine physiological aspects of the crofton weed that might render the plant vulnerable to environmental stress. Stresses imposed on crofton weed were heat （progressively increasing temperatures： 25 ℃, 30 ℃, 35 ℃, 38℃ and 42 ℃ at 24 h intervals）, cold （progressively decreasing temperatures： 25 ℃, 20 ℃, 15℃, 10 ℃ and 5℃ at 24h intervals）, and drought （without watering up to 4days）. The three stresses induced oxidative damage as evidenced by an increase in lipid peroxidation. The effect varied with the stress imposed and the length of exposure. The activity of superoxide dismutase （SOD） increased in response to all stresses but was not significantly different from the controls （P 〈 0.05） when exposed to cold stress. Catalase （CAT） activity decreased in response to heat and drought stress but increased when exposed to cold conditions. Guaiacol peroxidase （POD） and glutathione reductase （GR） activities increased in response to cold and drought but decreased in response to heat stress. The activity of ascorbata peroxidase （APX） responded differently to all three stresses. Monodehydroascorbate reductase （MDHAR） activity decreased in response to heat and drought, and slightly increased in response to the cold stress but was not significantly different from the controls （P 〈 0.05）. The activity of dehydroascorbata reductase （DHAR） increased in response to all three stresses. Taken together, the co-ordinate increase of the oxygen-detoxifying enzymes might be more effective to protect crofton weed from the accumulation of oxygen radicals at low temperatures rather than at high temperatures.     

Increased oxidative-modifications of cytosolic proteins in 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)-exposed rat liver

It is well established that 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) causes acute liver damage in animals and humans. The aim of this study was to identify and characterize oxidative modification and inactivation of cytosolic proteins in MDMA‐exposed rats. Markedly increased levels of oxidized and nitrated cytosolic proteins were detected 12 h after the second administration of two consecutive MDMA doses (10 mg/kg each). Comparative 2‐DE analysis showed markedly increased levels of biotin‐N‐methylimide‐labeled oxidized cytosolic proteins in MDMA‐exposed rats compared to vehicle‐treated rats. Proteins in the 22 gel spots of strong intensities were identified using MS/MS. The oxidatively modified proteins identified include anti‐oxidant defensive enzymes, a calcium‐binding protein, and proteins involved in metabolism of lipids, nitrogen, and carbohydrates (glycolysis). Cytosolic superoxide dismutase was oxidized and its activity significantly inhibited following MDMA exposure. Consistent with the oxidative inactivation of peroxiredoxin, MDMA activated c‐Jun N‐terminal protein kinase and p38 kinase. Since these protein kinases phosphorylate anti‐apoptotic Bcl‐2 protein, their activation may promote apoptosis in MDMA‐exposed tissues. Our results show for the first time that MDMA induces oxidative‐modification of many cytosolic proteins accompanied with increased oxidative stress and apoptosis, contributing to hepatic damage.     

Endogenous antioxidant defence system in rat liver following mercury chloride oral intoxication

Mercury is a highly toxic metal which induces oxidative stress. Superoxide dismutases, catalase, and glutathion peroxidase are proteins involved in the endogenous antioxidant defence system. In the present study rats were administered orally, by gavage, a single daily dose of HgCl2 for three consecutive days. In order to find a relation between the proteins involved in the antioxidant defence and mercury intoxication, parameters of liver injury, redox state of the cells, as well as intracellular protein levels and enzyme activities of Mn-dependent superoxide dismutase (MnSOD), Cu-Zn-dependent superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase (GPx) were assayed both in blood and in liver homogenates. HgCl2 at the doses of 0.1 mg/kg produced liver damage which that was detected by a slight increase in serum alanine aminotransferase and gamma glutamyl transferase. Hepatic GSH/GSSG ratio was assayed as a parameter of oxidative stress and a significant decrease was detected, as well as significant increases in enzyme activities and protein levels of hepatic antioxidant defence systems. Changes in both MnSOD and CuZnSOD were parallel to those of liver injury and oxidative stress, while the changes detected in catalase and GPx activities were progressively increased along with the mercury intoxication. Other enzyme activities related to the glutathione redox cycle, such as glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), also increased progressively. We conclude that against low doses of mercury that produce a slight oxidative stress and liver injury, the response of the liver was to induce the synthesis and activity of the enzymes involved in the endogenous antioxidant system. The activities of all the enzymes assayed showed a rapidly induced coordinated response.     

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label‐free quantitative shotgun proteomic analysis was performed. A total of 2042 non‐redundant proteins were identified from the five temperature points. Fifty‐five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold‐responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 ( http://proteomecentral.proteomexchange.org/dataset/PXD000977 ).     

Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish

Acute, short term cooling of North Sea eelpout Zoarces viviparus is associated with a reduction of tissue redox state and activation of hypoxia inducible factor (HIF-1) in the liver. The present study explores the response of HIF-1 to seasonal cold in Zoarces viviparus, and to latitudinal cold by comparing the eurythermal North Sea fish to stenothermal Antarctic eelpout (Pachycara brachycephalum). Hypoxic signalling (HIF-1 DNA binding activity) was studied in liver of summer and winter North Sea eelpout as well as of Antarctic eelpout at habitat temperature of 0°C and after long-term warming to 5°C. Biochemical parameters like tissue iron content, glutathione redox ratio, and oxidative stress indicators were analyzed to see whether the cellular redox state or reactive oxygen species formation and HIF activation in the fish correlate. HIF-1 DNA binding activity was significantly higher at cold temperature, both in the interspecific comparison, polar vs. temperate species, and when comparing winter and summer North Sea eelpout. Compared at the low acclimation temperatures (0°C for the polar and 6°C for the temperate eelpout) the polar fish showed lower levels of lipid peroxidation although the liver microsomal fraction turned out to be more susceptible to lipid radical formation. The level of radical scavenger, glutathione, was twofold higher in polar than in North Sea eelpout and also oxidised to over 50%. Under both conditions of cold exposure, latitudinal cold in the Antarctic and seasonal cold in the North Sea eelpout, the glutathione redox ratio was more oxidised when compared to the warmer condition. However, oxidative damage parameters (protein carbonyls and thiobarbituric acid reactive substances (TBARS) were elevated only during seasonal cold exposure in Z. viviparus. Obviously, Antarctic eelpout are keeping oxidative defence mechanisms high enough to avoid accumulation of oxidative damage products at low habitat temperature. The paper discusses how HIF could be instrumental in cold adaptation in fish.     

Wide-gene expression analysis of lipid-relevant genes in nutritionally challenged gilthead sea bream (Sparus aurata)

Disturbances of lipid metabolism are a major problem in livestock fish and the present study analysed the different tissue expression patterns and regulations of 40 lipid-relevant genes in gilthead sea bream. Nineteen sequences, including fatty acid elongases (4), phospholipases (7), acylglycerol lipases (8) and lipase-maturating enzymes (1), were new for gilthead sea bream (GenBank, JX975700, JX975701, JX975702, JX975703, JX975704, JX975705, JX975706, JX975707, JX975708, JX975709, JX975710, JX975711, JX975712, JX975713, JX975714, JX975715, JX975716, JX975717 and JX975718). Up to six different lipase-related enzymes were highly expressed in adipose tissue and liver, which also showed a high expression level of Δ6 and Δ9 desaturases. In the brain, the greatest gene expression level was achieved by the very long chain fatty acid elongation 1, along with relatively high levels of Δ9 desaturases and the phospholipase retinoic acid receptor responder. These two enzymes were also expressed at a high level in white skeletal muscle, which also shared a high expression of lipid oxidative enzymes. An overall down-regulation trend was observed in liver and adipose tissue in response to fasting following the depletion of lipid stores. The white skeletal muscle of fasted fish showed a strong down-regulation of Δ9 desaturases in conjunction with a consistent up-regulation of the “lipolytic machinery” including key enzymes of tissue fatty acid uptake and mitochondrial fatty acid transport and oxidation. In contrast, the gene expression profile of the brain remained almost unaltered in fasted fish, which highlights the different tissue plasticity of lipid-related genes. Taken together, these findings provide new fish genomic resources and contribute to define the most informative set of lipid-relevant genes for a given tissue and physiological condition in gilthead sea bream.     

Energy reserves and metabolic status affect the acclimation of gilthead sea bream (Sparus aurata) to cold

During winter, low temperatures induce a direct metabolic depression in gilthead sea bream, without any significant compensatory effect below 13 °C. The present study therefore focused on how to improve response to cold in these fish, looking specifically at the two factors of diet (high energy, HiE, and low energy, LoE) and activity (normal, ? SW, and sustained activity, + SW) prior to exposure to cold. Following a preparatory period of 75 days water was adjusted to 10 °C and kept for 40 days. Enzymatic activities and store deposition revealed that the HiE?SW group had acquired an energy surplus whilst the LoE+SW group exhibited an energy deficit. Liver enzyme activities evidenced diet dependence: LoE groups showed greater glucose-6-phosphate dehydrogenase activity and HiE groups showed greater lipoprotein lipase and hepatic lipase activities. Moreover, the HiE?SW group's lower citrate synthase/cytochrome-c-oxidase ratio reflected the energy surplus available. Perivisceral fat mobilisation caused by cold stress affected liver integrity, resulting in a pre-steatotic condition for the HE?SW group. The differences in liver enzyme activities produced by pre-cold conditions disappeared at low temperatures and enzymatic activities did not compensate. Therefore any improvement that would enable gilthead sea bream to face up to winter must be achieved prior to the appearance of low temperatures.     

Cloning and analysis of expression of a gilthead sea bream (Sparus aurata) Mx cDNA

In the current work, we have cloned and sequenced the full cDNA for a Mx protein in the gilthead sea bream (Sparus aurata) by RACE PCR. The Mx cDNA of 2182 bp contained an open reading frame of 1857 bp that codes for a protein of 618 aa. Within the coding sequence, characteristic features of Mx proteins were found, such as a tripartite guanosine-5'-triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD), the signature of the dynamin family, LPRG(S/K)GIVTR, and a sequence that codes for a leucine zipper at the C-terminal region of the protein. An RT-PCR was optimised to estimate the level of expression of Mx protein in sea bream. Through this method we determined that Mx is constitutively expressed in head kidney, liver, spleen, heart, gills, muscle and brain of healthy sea bream. Intramuscular challenge of sea bream with polyinosinic:polycytidylic acid (Poly I:C) up-regulated Mx expression in liver, head kidney, spleen and muscle. Constitutive expression was also found in isolated head kidney macrophages and blood leukocytes. This expression was significantly up-regulated by addition of Poly I:C. Mx was not constitutively expressed in the sea bream established cell line, SAF-1, but Poly I:C and nodavirus were also capable of inducing Mx expression in this cell line.     

Proteomic analysis of the cold stress response in the moss,Physcomitrella patens

Cold stress has adverse effects on plant growth and development. Plants respond and acclimate to cold stress through various biochemical and physiological processes, thereby acquiring stress tolerance. To better understand the basis for tolerance, we carried out a proteomic study in the model moss, Physcomitrella patens, characterizing gametophore proteins with 2‐DE and mass spectroscopy. Following exposure to 0°C for up to 3 days, out of the more than 1000 protein spots reproducibly resolved, only 45 changed in abundance by at least 1.5‐fold. Of these, 35 were identified by tryptic digestion and mass spectroscopy. Photosynthetic proteins decreased, whereas many catabolic proteins increased. In addition, cold stress up‐regulated a variety of signaling, cytoskeleton, and defense proteins and few proteins in these classes were down‐regulated. Up‐regulated proteins include the 14‐3‐3‐like protein, actin, HSP70s, lipoxygenases, and cytochrome P450 proteins. These results point to pathways that are important for the mechanism of cold stress response in P. patens and by extension to the entire plant kingdom.     

Responses of antioxidant enzymes to cold and high light are not correlated to freezing tolerance in natural accessions of Arabidopsis thaliana

Low temperatures and high light cause imbalances in primary and secondary reactions of photosynthesis, and thus can result in oxidative stress. Plants employ a range of low‐molecular weight antioxidants and antioxidant enzymes to prevent oxidative damage, and antioxidant defence is considered an important component of stress tolerance. To figure out whether oxidative stress and antioxidant defence are key factors defining the different cold acclimation capacities of natural accessions of the model plant Arabidopsis thaliana, we investigated hydrogen peroxide (H₂O₂) production, antioxidant enzyme activity and lipid peroxidation during a time course of cold treatment and exposure to high light in four differentially cold‐tolerant natural accessions of Arabidopsis (C24, Nd, Rsch, Te) that span the European distribution range of the species. All accessions except Rsch (from Russia) had elevated H₂O₂ in the cold, indicating that production of reactive oxygen species is part of the cold response in Arabidopsis. Glutathione reductase activity increased in all but Rsch, while ascorbate peroxidase and superoxide dismutase were unchanged and catalase decreased in all but Rsch. Under high light, the Scandinavian accession Te had elevated levels of H₂O₂. Te appeared most sensitive to oxidative stress, having higher malondialdehyde (MDA) levels in the cold and under high light, while only high light caused elevated MDA in the other accessions. Although the most freezing‐tolerant, Te had the highest sensitivity to oxidative stress. No correlation was found between freezing tolerance and activity of antioxidant enzymes in the four accessions investigated, arguing against a key role for antioxidant defence in the differential cold acclimation capacities of Arabidopsis accessions.