Cu-induced Oxidative Stress Research Articles

Insights into tolerance mechanisms of earthworms (Eisenia fetida) in copper-contaminated soils by integrating multi-omics analyses

Earthworms can resist high levels of soil copper (Cu) contamination and play an essential role in absorbing them effectively. However, the molecular mechanisms underlying Cu tolerance in earthworms are poorly understood. To address this research gap, we studied alterations of Eisenia fetida in antioxidant enzymes, gut microbiota, metabolites, and genes under varying levels of Cu exposure soils (0, 67.58, 168.96, 337.92 mg/kg). Our results revealed a reduction in antioxidant enzyme activities across all treatment groups, indicating an adaptive response to alleviate Cu-induced oxidative stress. Analysis of gut microbiota revealed a significant increase in the abundance of bacteria associated with nutrient uptake and Cu2+ excretion under Cu stress. Furthermore, metabolomic analysis discovered an increase in certain metabolites associated with energy metabolism, such as pyruvic acid, L-malic acid, and fumaric acid, as Cu concentration escalated. These results suggested that enhanced energy supply contributes to the elevated tolerance of E. fetida towards Cu. Additionally, transcriptome analysis not only identified crucial detoxification genes (Hsp70, CTSL, GST, CHAC, and GCLC), but also confirmed the critical role of glutathione metabolism as a key pathway in E. fetida Cu detoxification processes. These findings provide a new perspective on the molecular mechanisms of Cu tolerance in earthworms.

Nitrate reduces copper toxicity by preventing oxidative stress and inhibiting copper translocation from roots to shoots in Liriodendron Chinense.

Nitrogen forms can affect metal accumulation in plants and tolerance to metals, but a few published studies on the effects on Cu toxicity and Cu accumulation in plants are scarce. Thus, the objective of this study was to evaluate the responses of Liriodendron chinense to different nitrogen forms, by the oxidative stress, antioxidant enzymes system, GSH-AsA cycle, Cu uptake, translocation, and accumulation under Cu stress. We found that Cu-induced growth inhibiting was alleviated by added exclusive NO3--N. Adding N as NH4+-N with or without NO3--N was aggravated as evidenced by significantly elevated malonaldehyde (MDA) and hydrogen peroxide (H2O2) compared to N-Null. Cu exposure and adding NH4+-N inhibited superoxide dismutase activity, but remarkably stimulated the activities of catalase and peroxidase, the efficiency of glutathione-ascorbate (GSH-AsA) cycle, and the activity of glutathione reductase and nitrate reductase, with respect to the control. However, adding exclusive NO3--N progressively restored the alteration of antioxidant to prevent Cu-induced oxidative stress. Additionally, adding exclusive NO3--N significantly promoted the Cu uptake and accumulation in roots, but reduced Cu concentration in leaves, accompanied by the inhibited Cu translocation factor from roots to shoots by 36.7%, when compared with N-Null. Overall, adding NO3--N alleviated its Cu toxicity by preventing Cu-induced oxidative stress and inhibiting Cu translocation from roots to shoots, which provides an effective strategy for phytostabilization in Cu-contaminated lands.

Efficacious use of Micrococcus yunnanensis GKSM13 for the growth of rice seedlings under copper stress with elucidation into genomic traits

Rice is widely cultivated worldwide, and beneficial bactrial colonization are particularly desirable for sustainable agriculture because they promote growth and production by preventing excessive heavy metal contamination. The present study was conducted with the aim of improving the growth of rice seedlings (MTU1010 variety) under copper (Cu) stress by using Cu-tolerant plant growth promoting bacteria (PGPB) Micrococcus yunnanensis GKSM13. Strain GKSM13 was able to produce plant growth promoting factors (PGPFs) such as indole-3-acetic acid (IAA), gibberellin A3 (GA3) and ammonia, accumulate proline, fix N2, and inhibit 2,2-Diphenyl-1-picrylhydrazyl (DPPH). Rice seedlings treated with Cu2+ and co-inoculated with GKSM13 significantly improved their growth in morphological and biochemical aspects. When analysed by field emission scanning electron microscope (FE-SEM), GKSM13 was found to be associated with the root cells in the form of large number of coccoid cells. The uptake of Cu2+ in rice seedlings was reduced to 57.5% in the presence of GKSM13. Strain GKSM13 treatment also reduced Cu-induced oxidative stress of rice seedlings by activating antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX) and glutathione peroxidase (GPOX), which was supported by DPPH inhibition and reduction of malondialdehyde (MDA) accumulation. Insight into the genome of strain GKSM13 reveals the presence of tryptophan (trp), ent-kaurene, cyanase (cyn), phosphate-specific transport (pst), major facilitator superfamily transporter (MFS), sulphate transporter (cys), proline (pro) and SOD (sod) genes, which are responsible for promoting plant growth and alleviating Cu2+ stress. Therefore, the application of strain M. yunnanensis GKSM13 could provide a sustainable agricultural solution for Cu-affected mining areas.

Effect of arsenic and copper in kidney of mice: Crosstalk between Nrf2/ Keap1 pathway in apoptosis and pyroptosis

Arsenic (As) and copper (Cu) are two common contaminants in the environment. When organisms are exposed to As or/ and Cu in large quantities or for sustained periods, oxidative stress is induced, adversely affecting kidney function. However, the molecular mechanisms involved in As or/ and Cu-induced nephrotoxicity remain elusive. In this experiment, wild-type C57BL/6 and Nrf2-knockout mice (n = 24 each) were exposed to arsenic trioxide and copper chloride alone or in combination. Our research findings indicate that exposure to As or/ and Cu can activate the Nrf2 antioxidant pathway by upregulating the levels of Nrf2, HO-1, CAT, and downregulating the level of Keap1, thereby reducing As or/ and Cu-induced oxidative stress. Meanwhile, exposure induced kidney cell pyroptosis and apoptosis by promoting the expression of NLRP3 inflammasomes and Caspase-3, which peaked in mice co-treated with As and Cu. Subsequently, we investigated its role in As or/ and Cu-induced kidney injury by knocking out Nrf2. Our results show that after knocking out Nrf2, the expression of antioxidant factors CAT and HO-1 significantly decreased. Based on the low antioxidant capacity after Nrf2 knockout, the levels of NLRP3 inflammasome, GSDMD, and Caspase1 were significantly upregulated after exposure to As and Cu, indicating more severe cellular pyroptosis. In addition, the level of Caspase3-mediated apoptosis was also more severe. Taken together, there is crosstalk between Nrf2-mediated antioxidant capacity and apoptosis/ pyroptosis induced by exposure to As or/ and Cu. Depletion of Nrf2 alters its antioxidant capacity, ultimately leading to more severe apoptosis, pyroptosis, and nephrotoxicity.

Copper induced intestinal inflammation response through oxidative stress induced endoplasmic reticulum stress in Takifugu fasciatus

Copper (Cu) pollution in aquaculture water has seriously threatened the healthy and sustainable development of the aquaculture industry. Recently, many researchers have studied the toxic effects of Cu exposure on fish. However, the relationship between endoplasmic reticulum stress (ERS) and the inflammatory response, as well as its possible mechanisms, remain unclear. Particularly, information related to fish intestines must be expanded. Our study initially investigated the mechanisms underlying intestinal toxicity and inflammation resulting from Cu-induced ERS in vivo and in vitro in Takifugu fasciatus. In vivo study, T. fasciatus were treated with different concentrations (control, 20, and 100 µg/L) of Cu exposure for 28 days, causing intestinal oxidative stress, ERS, inflammatory responses, and histopathological and ultrastructural damage. Transcriptomic data further showed that Cu exposure caused ERS, as well as inflammatory responses, in the intestinal tracts of T. fasciatus. In vitro experiments on the intestinal cells of T. fasciatus showed that Cu exposure treatment (7.5 µg/mL) for 24 h induced ERS and increased mitochondrial numbers and inflammatory responses. In contrast, the addition of 4-phenylbutyric acid (4-PBA) alleviated ERS and inflammatory response in the Cu-exposed group. Furthermore, the reactive oxygen species (ROS) inhibitor, N-Acetyl-l-cysteine (NAC), effectively alleviated Cu-induced ERS. In conclusion, our in vivo and in vitro studies have confirmed that oxidative stress triggers the ERS pathway, which is involved in the intestinal inflammatory response. Our study provides new insights into the relationship among Cu-induced oxidative stress, ERS, and inflammatory responses in fish, as well as for the healthy culture of fish in aqueous environments.

Revisiting the pro-oxidant activity of copper: interplay of ascorbate, cysteine, and glutathione.

Copper (Cu) is essential for most organisms, but it can be poisonous in excess, through mechanisms such as protein aggregation, trans-metallation, and oxidative stress. The latter could implicate the formation of potentially harmful reactive oxygen species (O2•-, H2O2, and HO•) via the redox cycling between Cu(II)/Cu(I) states in the presence of dioxygen and physiological reducing agents such as ascorbate (AscH), cysteine (Cys), and the tripeptide glutathione (GSH). Although the reactivity of Cu with these reductants has been previously investigated, the reactions taking place in a more physiologically relevant mixture of these biomolecules are not known. Hence, we report here on the reactivity of Cu with binary and ternary mixtures of AscH, Cys, and GSH. By measuring AscH and thiol oxidation, as well as HO• formation, we show that Cu reacts preferentially with GSH and Cys, halting AscH oxidation and also HO• release. This could be explained by the formation of Cu-thiolate clusters with both GSH and, as we first demonstrate here, Cys. Moreover, we observed a remarkable acceleration of Cu-catalyzed GSH oxidation in the presence of Cys. We provide evidence that both thiol-disulfide exchange and the generated H2O2 contribute to this effect. Based on these findings, we speculate that Cu-induced oxidative stress may be mainly driven by GSH depletion and/or protein disulfide formation rather than by HO• and envision a synergistic effect of Cys on Cu toxicity.

ʟ-glutamic acid modulates antioxidant defense systems and nutrient homeostasis in lentil (Lens culinaris Medik.) under copper toxicity.

Copper (Cu), an essential micronutrient, can generate reactive oxygen species (ROS) at its supra-optimal level in living cells as a transition metal, thus producing oxidative stress in plants. Therefore, protecting plants from Cu-induced oxidative stress via the exogenous application of chemical substances, particularly L-glutamic acid (L-Glu), could be a viable strategy for mitigating the toxicity of Cu. The aim of our present study was to investigate how ʟ-Glu protects lentil seedlings from oxidative stress produced by toxic Cu and allows them to survive under Cu toxicity. The results exhibited that when lentil seedlings were exposed to excessive Cu, their growth was inhibited and their biomass decreased due to an increase in Cu accumulation and translocation to the root, shoot, and leaves. Exposure to toxic Cu also depleted photosynthetic pigments, imbalanced water content, and other essential nutrients, increased oxidative stress, and reduced enzymatic and non-enzymatic antioxidants. However, pre-treatment of ʟ-Glu improved the phenotypic appearance of lentil seedlings, which was distinctly evidenced by higher biomass production, maintenance of water balance, and an increase in photosynthetic pigments when exposed to toxic Cu. ʟ-Glu also protected the seedlings from Cu-induced oxidative stress by reducing the oxidative stress marker, specifically by the efficient action of enzymatic and non-enzymatic antioxidants, particularly ascorbate, catalase, monodehydroascorbate, and glutathione peroxidase and maintaining redox balance. Furthermore, ʟ-Glu assisted in maintaining the homeostasis of Cu and other nutrient in the roots, shoots, and leaves of lentil. Collectively, our results provide evidence of the mechanism of ʟ-Glu-mediated protective role in lentil against Cu toxicity, thus proposed as a potential chemical for managing Cu toxicity not only in lentil but also other plants.

Bacillus coagulans (Weizmannia coagulans) XY2 attenuates Cu-induced oxidative stress via DAF-16/FoxO and SKN-1/Nrf2 pathways and gut microbiota regulation

Copper (Cu) pollution has become a serious environmental problem especially in recent decades. In this study, the mechanisms of Bacillus coagulans (Weizmannia coagulans) XY2 against Cu-induced oxidative stress were explored through a dual model. In mice, Cu disturbed microbial community structure, revealing an increased level of Enterorhabdus abundance and decreased levels of Intestinimonas, Faecalibaculu, Ruminococcaceae and Coriobacteriaceae_UCG-002 abundance. Meanwhile, B. coagulans (W. coagulans) XY2 intervention reversed this trend along with alleviated Cu-induced metabolic disturbances by increasing levels of hypotaurine and L-glutamate and declining levels of phosphatidylcholine and phosphatidylethanolamine. In Caenorhabditis elegans, nuclear translocation of DAF-16 and SKN-1 was inhibited by Cu, which in turn suppressed antioxidant-related enzymes activities. XY2 mitigated biotoxicity associated with oxidative damage caused by Cu exposure via regulating DAF-16/FoxO and SKN-1/Nrf2 pathways and intestinal flora to eliminate excess ROS. Our study provides a theoretical basis formulating future strategy of probiotics against heavy metal contamination.

Alpha Lipoic Acid and Monoisoamyl-DMSA Combined Treatment Ameliorates Copper-Induced Neurobehavioral Deficits, Oxidative Stress, and Inflammation.

Copper (Cu), being an essential trace metal, plays several roles in biological processes, though exposure to Cu can be potentially toxic to the brain and a few other soft organs. In the present study, we investigated the effects of the combined administration of monoisoamyl 2, 3-dimercaptosuccinic acid (MiADMSA), which is a new chelator, and alpha lipoic acid (ALA) and an antioxidant that is made naturally in the body and is also found in foods, against Cu-induced oxidative stress in rats. Rats were exposed to 20 mg/kg copper sulfate for 16 weeks once a day via the oral route. After 16 weeks of exposure, animals were divided into different sub-groups. Group I was divided into three subgroups: Group IA, control; Group IB, MiADMSA (75 mg/kg, oral); Group IC, ALA (75 mg/kg, oral), while Group II was divided into four subgroups: Group IIA, Cu pre-exposed; Group IIB, Cu+ MiADMSA; Group IIC, Cu+ ALA; Group IID, Cu+ ALA+ MiADMSA. Exposure to Cu led to significant neurobehavioral abnormalities; treatment with MiADMSA, and in particular MiADMSA + ALA, significantly ameliorated the neurobehavioral parameters and restored the memory deficits in rats. Oxidative stress variables (ROS, nitrite, TBARS, SOD, catalase) and inflammatory markers (TNF-α, and IL-1β), which were altered on Cu exposed rats, also responded favorably to ALA+ MiADMSA combined treatment. Thus, combined administration of MiADMSA and ALA might be a better treatment strategy than monotherapy with MiADMSA or ALA against Cu-induced neurotoxicity, particularly in reducing oxidative stress, neurobehavioral abnormalities, and inflammatory markers.

Subcellular redox responses reveal different Cu-dependent antioxidant defenses between mitochondria and cytosol.

Excess intracellular Cu perturbs cellular redox balance and thus causes diseases. However, the relationship between cellular redox status and Cu homeostasis and how such an interplay is coordinated within cellular compartments has not yet been well established. Using combined approaches of organelle-specific redox sensor Grx1-roGFP2 and non-targeted proteomics, we investigate the real-time Cu-dependent antioxidant defenses of mitochondria and cytosol in live HEK293 cells. The Cu-dependent real-time imaging experiments show that CuCl2 treatment results in increased oxidative stress in both cytosol and mitochondria. In contrast, subsequent excess Cu removal by bathocuproine sulfonate, a Cu chelating reagent, lowers oxidative stress in mitochondria but causes even higher oxidative stress in the cytosol. The proteomic data reveal that several mitochondrial proteins, but not cytosolic ones, undergo significant abundance change under Cu treatments. The proteomic analysis also shows that proteins with significant changes are related to mitochondrial oxidative phosphorylation and glutathione synthesis. The differences in redox behaviors and protein profiles in different cellular compartments reveal distinct mitochondrial and cytosolic response mechanisms upon Cu-induced oxidative stress. These findings provide insights into how redox and Cu homeostasis interplay by modulating specific protein expressions at the subcellular levels, shedding light on understanding the effects of Cu-induced redox misregulation on the diseases.

Role of nuclear pregnane X receptor in Cu-induced lipid metabolism and xenobiotic responses in largemouth bass (Micropterus salmoides).

The pregnane X receptor (PXR) is a master xenobiotic-sensing receptor in response to toxic byproducts, as well as a key regulator in intermediary lipid metabolism. Therefore, the present study was conducted to investigate the potential role of PXR in mediating the lipid dysregulation and xenobiotic responses under Cu-induced stress in largemouth bass (Micropterus salmoides). Four groups of largemouth bass (52.66 ± 0.03 g) were treated with control, Cu waterborne (9.44 μmol/L), Cu+RIF (Rifampicin, 100 mg/kg, PXR activator), and Cu+KET (Ketoconazole, 20 mg/kg, PXR inhibitor) for 48 h. Results showed that Cu exposure significantly elevated the plasma stress indicators and triggered antioxidant systems to counteract Cu-induced oxidative stress. Acute Cu exposure caused liver steatosis, as indicated by the significantly higher levels of plasma triglycerides (TG), lipid droplets, and mRNA levels of lipogenesis genes in the liver. Liver injuries were detected, as shown by hepatocyte vacuolization and severe apoptotic signals after Cu exposure. Importantly, Cu exposure significantly stimulated mRNA levels of PXR, suggesting the response of this regulator in the xenobiotic response. The pharmacological intervention of PXR by the agonist and antagonist significantly altered hepatic mRNA levels of PXR, implying that RIF and KET were effective agents of PXR in largemouth bass. Administration of RIF significantly exacerbated liver steatosis, and such alterations were dependent on the regulations on pparγ and cd36 rather than srebp1 signaling, which suggested that PXR-PPARγ might be another pathway for Cu-induced lipid deposition in fish. Whereas, KET administration showed reverse effects on lipid metabolism as indicated by the lower hepatic TG levels, suppressed mRNA levels of pparγ and cd36. Activation of PXR stimulated autophagy and inhibited apoptosis, leading to lower hepatic vacuolization; while inhibition of PXR showed higher apoptotic signals, inhibition of autophagic genes and stimulation of apoptotic genes. Taken together, PXR played a cytoprotective role in Cu-induced hepatotoxicity through regulations on autophagy and apoptosis. Overall, our data has demonstrated for the first time on the dual roles of PXR as a co-regulator in mediating xenobiotic responses and lipid metabolism in fish, which implying the potential of PXR as a therapy target for xenobiotics-induced lipid dysregulation and hepatotoxicity.

Potential Use of Copper-Contaminated Soils for Hemp (Cannabis sativa L.) Cultivation

To mitigate climate change, reducing greenhouse gas emissions can be achieved by decreasing the use of fossil fuels and increasing that of alternative sources, such as energy crops. However, one of the most important problems in the use of biomass as a fuel is that of changing soil use and consumption, leading to competition with food crops. We addressed the topic by evaluating the possibility to exploit contaminated areas for energy crops cultivation. Indeed, soil contamination makes land inappropriate for cultivation, with damaging consequences for ecosystems, as well as posing serious health hazards to living beings. Specifically, this work aimed to evaluate the ability of hemp (Cannabis sativa L.) plants to grow on a copper (Cu)-contaminated medium. In addition, the effectiveness of an environment-friendly treatment with sulfate in improving plant ability to cope with Cu-induced oxidative stress was also explored. Results showed that plants were able to grow at high Cu concentrations. Therefore, hemp could represent an interesting energy crop in Cu-contaminated soils. Although the response of Cu-treated plants was evidenced by the increase in thiol content, following modulation of sulfur metabolism, it remains to be clarified whether the use of exogenous sulfate could be an agronomic practice to improve crop performance under these edaphic conditions.

Copper (Cu) induced changes of lipid metabolism through oxidative stress-mediated autophagy and Nrf2/PPARγ pathways

Oxidative stress can induce occurrence of non-alcoholic fatty liver disease (NAFLD). Nrf2 is a central regulator of cellular oxidative stress and also participates in the control of lipid deposition and metabolism. Here, we hypothesize that oxidative stress-mediated Nrf2 activation participates in the regulation of the Cu-induced lipid deposition. We found that Cu excess activated oxidative stress and autophagy, up-regulated lipogenesis and lipid metabolism, suppressed Keap1 expression and activated Nrf2 signaling. Moreover, Cu induced lipid deposition via oxidative stress and the mitochondrial dysfunction. Oxidative stress mediated Cu-induced activation of Nrf2 and autophagy. The activation of autophagy helps to alleviate Cu-induced lipid deposition and accordingly provided a protective role against Cu-induced NAFLD. Meantime, Cu-induced oxidative stress promoted Nrf2 recruitment to the PPARγ promoter, inducing target gene transcription, and subsequent lipogenesis. Our findings, for the first time, provide direct evidences for Nrf2 function in the modulation of lipogenic metabolism via the transcriptional activation of PPARγ, and elucidate the mechanisms by which Nrf2 functions as the central regulator of lipogenic genes and highlights the significance of Nrf2 as potential therapeutic targets for oxidative stress-associated obesity and NAFLD for fish and human beings.

Modeling Alleviative Effects of Ca, Mg, and K on Cu-Induced Oxidative Stress in Grapevine Roots Grown Hydroponically.

The aim of this study was to determine the pattern of alleviation effects of calcium (Ca), magnesium (Mg), and potassium (K) on copper (Cu)-induced oxidative toxicity in grapevine roots. Root growth, Cu and cation accumulation, reactive oxygen species (ROS) production, and antioxidant activities were examined in grapevine roots grown in nutrient solutions. The experimental setting was divided into three sets; each set contained a check (Hoagland solution only) and four treatments of simultaneous exposure to 15 μM Cu with four cation levels (i.e., Ca set: 0.5, 2.5, 5, and 10 mM Ca; Mg set: 0.2, 2, 4, and 8 mM Mg; K set: 0.6, 2.4, 4.8, and 9.6 mM K). A damage assessment model (DAM)-based approach was then developed to construct the dose-effect relationship between cation levels and the alleviation effects on Cu-induced oxidative stress. Model parameterization was performed by fitting the model to the experimental data using a nonlinear regression estimation. All data were analyzed by a one-way analysis of variance (ANOVA), followed by multiple comparisons using the least significant difference (LSD) test. The results showed that significant inhibitory effects on the elongation of roots occurred in grapevine roots treated with 15 μM Cu. The addition of Ca and Mg significantly mitigated phytotoxicity in root growth, whereas no significant effect of K treatment on root growth was found. With respect to oxidative stress, ROS and malondialdehyde (MDA) contents, as well as antioxidant (superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)) activities, were stimulated in the roots after exposure to 15 μM Cu for three days. Moreover, H2O2 levels decreased significantly as Ca, Mg, and K concentrations increased, indicating that the coexistence of these cations effectively alleviated Cu-induced oxidative stress; however, alleviative effects were not observed in the assessment of the MDA content and antioxidant enzyme activities. Based on the DAM, an exponential decay equation was developed and successfully applied to characterize the alleviative effects of Ca, Mg, and K on the H2O2 content induced by Cu in the roots. In addition, compared with Mg and K, Ca was the most effective cation in the alleviation of Cu-induced ROS. Based on the results, it could be concluded that Cu inhibited root growth and Ca and Mg absorption in grapevines, and stimulated the production of ROS, lipid peroxidation, and antioxidant enzymes. Furthermore, the alleviation effects of cations on Cu-induced ROS were well described by the DAM-based approach developed in the present study.

Activation of the ROS/HO-1/NQO1 signaling pathway contributes to the copper-induced oxidative stress and autophagy in duck renal tubular epithelial cells

The aim of this study was to investigate the crosstalk between oxidative stress and autophagy through the ROS/HO-1/NQO1 pathway caused by copper (Cu). Duck renal tubular epithelial cells were treated in Cu sulfate (CuSO4) (0, 100 and 200 μM) for 12 h, and in the combination of CuSO4 (200 μM) and reactive oxygen species (ROS) scavenger (butyl hydroxyanisole, BHA, 100 μM), or HO-1 inhibitor (zinc protoporphyrin, ZnPP, 10 μM) for 12 h. Results revealed that Cu could significantly elevate the levels of intracellular ROS, superoxide dismutase, hydrogen peroxide, malondialdehyde, glutathione, simultaneously reduce catalase and glutathione peroxidase levels, and upregulate HO-1, SOD-1, CAT, NQO1, GCLM mRNA levels and HO-1, SOD-1 protein levels. Additionally, Cu could observably increase the number of autophagosomes, acidic vesicle organelles (AVOs) and LC3 puncta; upregulate mRNA levels of mTOR, Beclin-1, ATG7, ATG5, ATG3, LC3II and protein levels of Beclin-1, LC3II/LC3I, downregulate LC3I mRNA level. Both treatments with BHA and ZnPP could significantly alleviate the changes of antioxidant indexes levels and ROS accumulation, reduce the increase of the number of autophagosomes, AVOs and LC3 puncta, and mitigate the above changed oxidative stress and autophagy related mRNA and protein levels induced by Cu. In summary, our findings indicated that excessive Cu could induce oxidative stress and autophagy by activating the ROS/HO-1/NQO1 pathway, and inhibition of HO-1 might attenuate Cu-induced oxidative stress and autophagy in duck renal tubular epithelial cells.

Sestrin2 Phosphorylation by ULK1 Induces Autophagic Degradation of Mitochondria Damaged by Copper-Induced Oxidative Stress.

Selective autolysosomal degradation of damaged mitochondria, also called mitophagy, is an indispensable process for maintaining integrity and homeostasis of mitochondria. One well-established mechanism mediating selective removal of mitochondria under relatively mild mitochondria-depolarizing stress is PINK1-Parkin-mediated or ubiquitin-dependent mitophagy. However, additional mechanisms such as LC3-mediated or ubiquitin-independent mitophagy induction by heavy environmental stress exist and remain poorly understood. The present study unravels a novel role of stress-inducible protein Sestrin2 in degradation of mitochondria damaged by transition metal stress. By utilizing proteomic methods and studies in cell culture and rodent models, we identify autophagy kinase ULK1-mediated phosphorylation sites of Sestrin2 and demonstrate Sestrin2 association with mitochondria adaptor proteins in HEK293 cells. We show that Ser-73 and Ser-254 residues of Sestrin2 are phosphorylated by ULK1, and a pool of Sestrin2 is strongly associated with mitochondrial ATP5A in response to Cu-induced oxidative stress. Subsequently, this interaction promotes association with LC3-coated autolysosomes to induce degradation of mitochondria damaged by Cu-induced ROS. Treatment of cells with antioxidants or a Cu chelator significantly reduces Sestrin2 association with mitochondria. These results highlight the ULK1-Sestrin2 pathway as a novel stress-sensing mechanism that can rapidly induce autophagic degradation of mitochondria under severe heavy metal stress.

Dynamic proteomic analysis of Phanerochaete chrysosporium under copper stress.

The model white rot fungus Phanerochaete chrysosporium is frequently preferred for heavy metal accumulation studies due to its high resistance to heavy metals, including copper (Cu). Here, the response of P. chrysosporium under Cu stress at different time points was investigated for the first time by a detailed proteomic analysis using 2DE MALDI-TOF/MS and nanoLC-MS/MS techniques. A total of 123 Cu-responsive protein spots were determined using 2DE approach, and 104 of them were corresponded to 73 distinct open reading frames (ORFs). Of identified ones, 88 spots were over-, and 16 spots were underrepresented. The majority of these proteins showed to the strongest response at 8th h of Cu exposure. Using nanoLC-MS/MS analysis, a total of 167 differentially produced proteins were identified from Cu-exposed cultures after enrichment of the membrane proteins followed by SILAC. Seventy four, 66, and 69 overrepresented, and 56, 71, and 64 underrepresented proteins were identified at 2h, 4h, and 8h of Cu exposure, respectively. The bioinformatic analysis of these proteins revealed that intracellular trafficking proteins such as Ran GTPase and a p24 family protein, and certain proteins involved in posttranslational modification, protein turnover and folding were Cu-responsive. Three important transcription factors (TFs), NAC, BTF3, and homeobox TFs, 40S and 60S ribosomal proteins, chaperones such as Hsp26/Hsp42 and mortalin, as well as 20S proteasome, 14-3-3 proteins and Hsp90 involve in Cu-stress response of P. chrysosporium. Moreover, certain elements of translation machinery, the proteins related with aspartate, methionine, and pyruvate metabolisms, transketolase, and trehalase related with carbohydrate metabolism, citrate synthase, fumarase, V-ATPase, and F0F1-type ATPase playing role in energy production and conversion, transport proteins such as multidrug resistance and p24 family proteins as well as actin-related proteins involved in cytoskeleton remodeling were determined to be Cu-responsive. The present proteome analysis revealed that P. chrysosporium mainly regulates translational and posttranslational processes, certain transport processes, many metabolic pathways and cytoskeleton to overcome the Cu-induced oxidative stress.

Effect of Citric Acid on Growth, Ecophysiology, Chloroplast Ultrastructure, and Phytoremediation Potential of Jute (Corchorus capsularis L.) Seedlings Exposed to Copper Stress.

Soil and water contamination from heavy metals and metalloids is one of the most discussed and caused adverse effects on food safety and marketability, crop growth due to phytotoxicity, and environmental health of soil organisms. A hydroponic investigation was executed to evaluate the influence of citric acid (CA) on copper (Cu) phytoextraction potential of jute (Corchorus capsularis L.). Three-weeks-old seedlings of C. capsularis were exposed to different Cu concentrations (0, 50, and 100 μM) with or without the application of CA (2 mM) in a nutrient growth medium. The results revealed that exposure of various levels of Cu by 50 and 100 μM significantly (p < 0.05) reduced plant growth, biomass, chlorophyll contents, gaseous exchange attributes, and damaged ultra-structure of chloroplast in C. capsularis seedlings. Furthermore, Cu toxicity also enhanced the production of malondialdehyde (MDA) which indicated the Cu-induced oxidative damage in the leaves of C. capsularis seedlings. Increasing the level of Cu in the nutrient solution significantly increased Cu uptake by the roots and shoots of C. capsularis seedlings. The application of CA into the nutrient medium significantly alleviated Cu phytotoxicity effects on C. capsularis seedlings as seen by plant growth and biomass, chlorophyll contents, gaseous exchange attributes, and ultra-structure of chloroplast. Moreover, CA supplementation also alleviated Cu-induced oxidative stress by reducing the contents of MDA. In addition, application of CA is helpful in increasing phytoremediation potential of the plant by increasing Cu concentration in the roots and shoots of the plants which is manifested by increasing the values of bioaccumulation (BAF) and translocation factors (TF) also. These observations depicted that application of CA could be a useful approach to assist Cu phytoextraction and stress tolerance against Cu in C. capsularis seedlings grown in Cu contaminated sites.

Comparative analyses of oxidative stress response and metallothionein induction in white sturgeon and rainbow trout during acute waterborne copper exposure

Early life-stages of the endangered white sturgeon (Acipenser transmontanus) have been shown to be among the most sensitive fishes to aqueous copper (Cu) exposure. In a recent analogous study, we examined the role of whole-body Cu accumulation and Na homeostasis in species-specific differences between the sensitivity of white sturgeon and a common laboratory fish model, rainbow trout, to Cu. However, the potential roles of important mechanisms such as Cu-induced oxidative stress and/or metallothionein (MT) induction as potential drivers of sensitivity of white sturgeon to Cu have not been investigated to date. Here, rainbow trout and white sturgeon from three different early life-stages were exposed to waterborne Cu for 96 h, following which major antioxidant parameters, lipid peroxidation and MT gene expression were evaluated. Results indicated that during larval and swim-up life-stages, Cu induced oxidative damage in white sturgeon was greater than in rainbow trout. Moreover, baseline glutathione (GSH) was significantly greater in rainbow trout than white sturgeon. Observations also suggested that trout exceedingly relied on GSH to combat Cu-induced oxidative stress as they grew older. In contrast, sturgeon recruited an increasing level of MT to neutralize Cu-induced oxidative stress and/or Cu loading. In our recent study, we demonstrated that Na homeostasis is more susceptible to Cu in white sturgeon than in rainbow trout. Collectively, these findings indicate that the greater degree of oxidative damage in early life-stages, in addition to the higher magnitude of the disruption of Na homeostasis, contributes to the higher sensitivity of white sturgeon to Cu exposure.

Appraising growth, oxidative stress and copper phytoextraction potential of flax (Linum usitatissimum L.) grown in soil differentially spiked with copper

Flax (Linum usitatissimum L.) is one of the oldest predominant industrial crops grown for seed, oil and fiber. The present study was executed to evaluate the morpho-physiological traits, biochemical responses, gas exchange parameters and phytoextraction potential of flax raised in differentially copper (Cu) spiked soil viz (0, 200, 400 and 600 mg Cu kg−1 soil) under greenhouse pot experiment. The results revealed that flax plants were able to grow up to 400 mg kg−1 Cu level without showing significant growth inhabitation while, further inference of Cu (600 mg kg−1) in the soil prominently inhibited flax growth and biomass accumulation. Compared to the control, contents of proline and malondialdehyde (MDA) were increased by 160.0% and 754.1% accordingly, at 600 mg Cu kg−1 soil level. The Cu-induced oxidative stress was minimized by the enhanced activities of superoxide dismutase (SOD) by 189.2% and guaiacol peroxidase (POD) by 300.8% in the leaves of flax at 600 mg Cu kg−1 soil level, compared to the untreated control. The plant Cu concentration was determined at 35, 70, 105 and 140 days after sowing (DAS) and results depicted that 16.9 times higher Cu concentration was accumulated in flax roots while little (14.9 times) was transported to the shoots at early stage of growth, i.e. 35 DAS. While at 140 DAS, Cu was highly (21.7 times) transported to the shoots while, only 12.3 times Cu was accumulated in the roots at 600 mg Cu kg−1 soil level, compared to control. Meanwhile, Cu uptake by flax was boosted up to 253 mg kg−1 from the soil and thereby extracted 43%, 39% and 41% of Cu at 200, 400 and 600 mg Cu kg−1 soil level, compared to initial Cu concentration. Therefore, study concluded that flax has a great potential to accumulate high concentration of Cu in its shoots and can be utilized as phytoremediation material when grown in Cu contaminated soils.