The aim of this study was to test the phytotoxicity and mode of action of bisphenol A (BPA) on
using a multibiomarker approach.
roots were exposed to BPA in concentration range 0-50 mg L
for 3 days. ...BPA even in the lowest applied concentration (1 mg L
) reduced root length, root fresh weight, and mitotic index. Additionally, the lowest BPA concentration (1 mg L
) decreased the level of gibberellic acid (GA
) in root cells. BPA at concentration 5 mg L
increased production of reactive oxygen species (ROS) that was followed by increase in oxidative damage to cells' lipids and proteins and activity of enzyme superoxide dismutase. BPA in higher concentrations (25 and 50 mg L
) induced genome damage detected as an increase in micronucleus (MNs) and nuclear buds (NBUDs). BPA at >25 mg L
induced synthesis of phytochemicals. Results of this study using multibiomarker approach indicate that BPA is phytotoxic to
roots and has shown genotoxic potential to plants, thus its presence in the environment should be monitored.
Copper ion-catalyzed oxidation of yeast SOD1 (ySOD1) was examined to determine early oxidative modifications, including oxidation of a crucial disulfide bond, and the structural and functional ...repercussions of these events. The study used distinct oxidative conditions: Cu
2+
/H
2
O
2
, Cu
2+
/H
2
O
2
/AscH
−
and Cu
2+
/H
2
O
2
/glucose. Capillary electrophoresis experiments and quantification of protein carbonyls indicate that ySOD1 is highly susceptible to oxidative modification and that changes can be detected within 0.1 min of the initiation of the reaction. Oxidation-induced structural perturbations, characterized by circular dichroism, revealed the formation of partially-unfolded ySOD1 species in a dose-dependent manner. Consistent with these structural changes, pyrogallol assay indicates a partial loss of enzymatic activity. ESI-MS analyses showed seven distinct oxidized ySOD1 species under mild oxidation within 0.1 min. LC/MS analysis after proteolytic digestion demonstrated that the copper-coordinating active site histidine residues, His47 and His49, were converted into 2-oxo-histidine. Furthermore, the Cu and Zn bridging residue, His64 is converted into aspartate/asparagine. Importantly, the disulfide-bond Cys58-Cys147 which is critical for the structural and functional integrity of ySOD1 was detected as being oxidized at Cys147. We propose, based on LC/MS analyses, that disulfide-bond oxidation occurs without disulfide bond cleavage. Modifications were also detected at Met85 and five surface-exposed Lys residues. Based on these data we propose that the Cys58-Cys147 bond may act as a sacrificial target for oxidants and protect ySOD1 from oxidative inactivation arising from exposure to Cu
2+
/H
2
O
2
and auto-inactivation during extended enzymatic turnover.
Image 1
•
Oxidation of yeast superoxide dismutase (ySOD1) by Cu
2+
/H
2
O
2
is examined.
•
Rapid modification of His, Met, Cys and Lys residues detected by LC-MS methods.
•
Oxidation of active site His residues and partial protein unfolding are early events.
•
The Cys58-Cys147 disulfide bond is oxidized and may act as a sacrificial target.
•
Excess exogenous Cu
2+
decreases protein damage and can reverse loss of activity.
Increased protein carbonyl content is a hallmark of cellular and organismal aging. Protein damage leading to the formation of carbonyl groups derives from direct oxidation of several amino acid side ...chains but can also derive through protein adducts formation with lipid peroxidation products and dicarbonyl glycating compounds. All these modifications have been implicated during oxidative stress, aging and age-related diseases. However, in most cases, the proteins targeted by these deleterious modifications as well as their consequences have not yet been clearly identified. Indeed, this is essential to determine whether and how these modified proteins are impacting on cellular function, on the development of the senescent phenotype and the pathogenesis of age-related diseases. In this context, protein modifications occurring during aging and upon oxidative stress as well as main proteomic methods for detecting, quantifying and identifying oxidized proteins are described. Relevant proteomics studies aimed at monitoring the extent of protein carbonylation and identifying the targeted proteins in the context of aging and oxidative stress are also presented. Proteomics approaches, i.e. fluorescent based 2D-gel electrophoresis and mass spectrometry methods, represent powerful tools for monitoring at the proteome level the extent of protein oxidative and related modifications and for identifying the targeted proteins.
Accumulation of damaged macromolecules, including oxidatively damaged (carbonylated) proteins, is a hallmark of cellular and organismal aging. Since protein carbonyls are the most commonly used markers of protein oxidation, different methods have been developed for the detection and quantification of carbonylated proteins. The identification of these protein targets is of valuable interest in order to understand the mechanisms by which damaged proteins accumulate and potentially affect cellular functions during oxidative stress, cellular senescence and/or aging in vivo. The specificity of hydrazide derivatives to carbonyl groups and the presence of a wide range of functional groups coupled to the hydrazide, allowed the design of novel strategies for the detection and quantification of carbonylated proteins. Of note is the importance of fluorescent probes for monitoring carbonylated proteins. Proteomics approaches, i.e. fluorescent based 2D-gel electrophoresis and mass spectrometry methods, represent powerful tools for monitoring at the proteome level the extent of protein oxidative and related modifications and for identifying the targeted proteins. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.
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•Protein carbonyl is a well recognized marker of protein oxidative damage.•Increased protein carbonyl content is a hallmark of cellular and organismal aging.•Oxidative damage targets specific proteins upon oxidative stress, cellular senescence and aging.•Detection, quantification and identification of carbonylated proteins can be achieved using fluorescent based proteomic methods.
Buildups of ammonia can cause potentially fatal brain swelling in mammals, but such swelling is reversible in the anoxia- and ammonia-tolerant goldfish (Carassius auratus). We investigated brain ...swelling and its possible relationship to oxidative stress in the brain and liver of goldfish acutely exposed to high external ammonia (HEA; 5 mmol/l NH
Cl) at two different acclimation temperatures (14°C, 4°C). Exposure to HEA at 14°C for 72h resulted in increased internal ammonia and glutamine concentrations in the brain, and it caused cellular oxidative damage in the brain and liver. However, oxidative damage was most pronounced in brain, in which there was a twofold increase in thiobarbituric acid-reactive substances, a threefold increase in protein carbonylation, and a 20% increase in water volume (indicative of brain swelling). Increased activities of catalase, glutathione peroxidase, and glutathione reductase in the brain suggested that goldfish upregulate their antioxidant capacity to partially offset oxidative stress during hyperammonemia at 14°C. Notably, acclimation to colder (4°C) water completely attenuated the oxidative stress response to HEA in both tissues, and there was no change in brain water volume despite similar increases in internal ammonia. We suggest that ammonia-induced oxidative stress may be responsible for the swelling of goldfish brain during HEA, but further studies are needed to establish a mechanistic link between reactive oxygen species production and brain swelling. Nevertheless, a high capacity to withstand oxidative stress in response to variations in internal ammonia likely explains why goldfish are more resilient to this stressor than most other vertebrates.
Hibernation confers resistance to ischemia–reperfusion injury in tissue, but the underlying mechanisms remain unclear. Suppression of mitochondrial respiration during torpor may contribute to this ...tolerance. To explore this concept, we subjected isolated liver mitochondria from torpid, interbout euthermic (IBE) and summer 13-lined ground squirrels (
Ictidomys tridecemlineatus
) to 5 min of anoxia, followed by reoxygenation (A/R). We also included rat liver mitochondria as a non-hibernating comparison group. Maximum respiration rates of mitochondria from torpid ground squirrels were not affected by A/R, but in IBE and summer, these rates decreased by 50% following A/R and in rats they decreased by 80%. Comparing net ROS production rates among groups, revealed seasonal differences; mitochondria from IBE and torpor produced 75% less ROS than summer ground squirrels and rats. Measurements of oxidative damage to these mitochondria, both freshly isolated, as well as pre- and post-A/R, demonstrated elevated damage to protein, but not lipids, in all groups. Hibernation likely generates oxidative stress, as freshly isolated mitochondria had greater protein damage in torpor and IBE than in summer and rats. When comparing markers of damage pre- and post-A/R, we found that when RET was active, rat macromolecules were more damaged than when RET is inhibited, but in TLGS markers of damage were similar. This result suggests that suppression of RET during hibernation, both in torpor and IBE, lessens oxidative stress produced during arousal. Taken together our study suggests that ischemia–reperfusion tolerance at the mitochondrial level is associated with metabolically suppressed oxidative phosphorylation during hibernation.
Cytotoxic compounds like reactive carbonyl compounds such as methylglyoxal (MG), melandialdehyde (MDA), besides the ROS accumulate significantly at higher levels under salinity stress conditions and ...affect lipids and proteins that inhibit plant growth and productivity. The detoxification of these cytotoxic compounds by overexpression of NADPH-dependent Aldo-ketoreductase (AKR1) enzyme enhances the salinity stress tolerance in tobacco. The PsAKR1 overexpression plants showed higher survival and chlorophyll content and reduced MDA, H2O2, and MG levels under NaCl stress. The transgenic plants showed reduced levels of Na+ levels in both root and shoot due to reduced reactive carbonyl compounds (RCCs) and showed enhanced membrane stability resulted in higher root growth and biomass. The increased levels of antioxidant glutathione and enhanced activity of superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) suggest AKR1 could protect these enzymes from the RCC induced protein carbonylation by detoxification process. The transgenics also showed higher activity of delta 1-pyrroline-5- carboxylate synthase (P5CS) enzyme resulted in increasedproline levels to maintain osmotic homeostasis. The results demonstrates that the AKR1 protects proteins or enzymes that are involved in scavenging of cytotoxic compounds by detoxifying RCCs generated under salinity stress.
•Salinity stress induces reactive cytotoxic compounds that affect plant growth.•Overexpression of Aldo-keto reductases detoxify RCCs and improve tolerance.•The AKR1 protects scavenging enzymes from protein carbonylation.•Protection of P5CS enzyme by AKR1 from carbonyl stress improve proline levels.•AKR1 protect chlorophyll degradation by ROS and RCC under salinity stress.
Previous studies on metal–ion catalyzed oxidation of α–synuclein oxidation have mostly used conditions that result in extensive modification precluding an understanding of the early events in this ...process. In this study, we have examined time-dependent oxidative events related to α–synuclein modification using six different molar ratios of Cu2+/H2O2/protein and Cu2+/H2O2/ascorbate/protein resulting in mild to moderate extents of oxidation. For a Cu2+/H2O2/protein molar ratio of 2.3:7.8:1 only low levels of carbonyls were detected (0.078 carbonyls per protein), whereas a molar ratio of 4.7:15.6:1 gave 0.22 carbonyls per α–synuclein within 15 min. With the latter conditions, rapid conversion of 3 out of 4 methionines (Met) to methionine sulfoxide, and 2 out of 4 tyrosines (Tyr) were converted to products including inter– and intra–molecular dityrosine cross-links and protein oligomers, as determined by SDS–PAGE and Western blot analysis. Limited histidine (His) modification was observed. The rapid formation of dityrosine cross-links was confirmed by fluorescence and mass–spectrometry. These data indicate that Met and Tyr oxidation are early events in Cu2+/H2O2-mediated damage, with carbonyl formation being a minor process. With the Cu2+/H2O2/ascorbate system, rapid protein carbonyl formation was detected with the first 5 min, but after this time point, little additional carbonyl formation was detected. With this system, lower levels of Met and Tyr oxidation were detected (2 Met and 1 Tyr modified with a Cu2+/H2O2/ascorbate/protein ratio of 2.3:7.8:7.8:1), but greater His oxidation. Only low levels of intra– dityrosine cross-links and no inter– dityrosine oligomers were detected under these conditions, suggesting that ascorbate limits Cu2+/H2O2-induced α–synuclein modification.
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•Met and Tyr oxidation are early and rapid events in Cu2+/H2O2– mediated damage to α–synuclein.•Met sulfoxide and dityrosine detected within 5 min of Cu2+/H2O2 exposure to α–synuclein.•Carbonyl formation is a minor process in early Cu2+/H2O2– mediated damage to α–synuclein.•Ascorbate limits Met and Tyr oxidation in Cu2+/H2O2/AscH-–mediated damage to α–synuclein.
Purpose: Oxidative stress (OS) has been implicated in the pathogenesis of metabolic syndrome (MetS). The acute change in OS biomarkers due to exercise, known as exercise-induced OS (EIOS), is ...postulated to be a more appropriate marker of OS compared to spot OS measures. These studies objectives were to investigate EIOS in participants with MetS and compare the associations between EIOS, spot OS measures and MetS severity.
Methods: Sixty-three participants with MetS had MetS severity assessed using the MetS Z-score. Participants undertook a cardiorespiratory fitness test (
O
2
peak) to volitional exhaustion (∼8-12 minutes). Plasma OS (total F2-isoprostanes (IsoP), protein carbonyls (PCs)) and antioxidant (glutathione peroxidase (GPx), total antioxidant status (TAS)) biomarkers were measured from samples obtained before and five minutes post-
O
2
peak test. Wilcoxon's signed-rank tests were used to determine changes in OS markers.
Results: There were no significant (p > 0.05) changes in OS or antioxidant biomarkers from pre- to post-exercise (median (interquartile range): IsoP -15.5 (-71.8 to 47.8) pg/mL; PC -0.01 (-0.16 to 0.13) nmol/mg protein; GPx 0.76 (-4.94 to 9.82) U/L, TAS 0.03 (0.00-0.05) mmol/L).
Conclusions: A
O
2
peak test to exhaustion failed to induce OS in participants with MetS. There were no associations between MetS severity and spot OS or EIOS biomarkers.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
In order to develop an understanding of the role of adjuvants in a popular glyphosate-based herbicide – Roundup® Concentrate Plus (RCP), on non-target organisms, the effects of pure glyphosate ...N-(phosphonomethyl)-glycine, RCP and a non-ionic surfactant – polyethoxylated tallowamine (POEA) were studied in the fruit fly Drosophila melanogaster. Acute exposure to sub-lethal concentrations of RCP (15 μg/mL) and POEA (45 μg/mL) reduced (p < 0.001) lifespan of female flies compared to untreated controls or glyphosate (100 μg/mL). Negative geotaxis responses in female flies were reduced (p < 0.05) following acute exposure to sub-lethal concentrations of RCP and POEA whereas glyphosate did not significantly affect this response compared to untreated flies. Acute exposure to sub-lethal concentrations of RCP and POEA elevated (p < 0.05) protein carbonyl levels while markedly (p < 0.01) inhibiting carbonyl reductase activity whereas glyphosate treatment did not significantly affect protein carbonyl levels or carbonyl reductase activity. Fecundity was reduced (p < 0.05) following exposure to sub-lethal concentrations of RCP and POEA whereas glyphosate did not affect fecundity. In vitro treatment of ovarian stem sheath (OSS) cells with sub-lethal concentrations of RCP and POEA revealed decreased cell viability and enhanced caspase activity indicative of pro-apoptotic processes after 48 h compared to untreated controls. Glyphosate however was non-toxic at the concentration used. The results suggest that RCP and the surfactant POEA are more toxic than pure glyphosate and inhibit fecundity in Drosophila by impairing cell viability through enhanced apoptosis.
•Toxicity of glyphosate, Roundup and POEA was tested on Drosophila melanogaster.•Roundup and POEA reduced lifespan, negative geotaxis and fecundity.•Glyphosate did not exhibit the deleterious effects of Roundup or POEA.•Roundup and POEA but not glyphosate were pro-apoptotic to ovarian cell cultures.
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