Boron-mediated amelioration of copper toxicity in Citrus sinensis seedlings involved reduced concentrations of copper in leaves and roots and their cell walls rather than increased copper fractions in their cell walls.

Little information is available on how boron (B) supplementation affects plant cell wall (CW) remodeling under copper (Cu) excess. 'Xuegan' (Citrus sinensis) seedlings were submitted to 0.5 or 350 µM Cu × 2.5 or 25 µM B for 24 weeks. Thereafter, we determined the concentrations of CW materials (CWMs) and CW components (CWCs), the degree of pectin methylation (DPM), and the pectin methylesterase (PME) activities and PME gene expression levels in leaves and roots, as well as the Cu concentrations in leaves and roots and their CWMs (CWCs). Additionally, we analyzed the Fourier transform infrared (FTIR) and X-ray diffraction (XRD) spectra of leaf and root CWMs. Our findings suggested that adding B reduced the impairment of Cu excess to CWs by reducing the Cu concentrations in leaves and roots and their CWMs and maintaining the stability of CWs, thereby improving leaf and root growth. Cu excess increased the Cu fractions in leaf and root pectin by decreasing DPM due to increased PME activities, thereby contributing to citrus Cu tolerance. FTIR and XRD indicated that the functional groups of the CW pectin, hemicellulose, cellulose, and lignin could bind and immobilize Cu, thereby reducing Cu cytotoxicity in leaves and roots.

Antagonistic Effect of Zinc Oxide Nanoparticles Dietary Supplementation Against Chronic Copper Waterborne Exposure on Growth, Behavioral, Biochemical, and Gene Expression Alterations of African Catfish, Clarias gariepinus (Burchell, 1822).

The harmful impact of waterborne copper (Cu) as a common abiotic stressor in aquatic environments has gained much more interest. The present study aimed to investigate the utilization of zinc oxide nanoparticles (ZnONPs) dietary supplementation to mitigate the chronic toxicity of Cu in African catfish (Clarias gariepinus). Two hundred and forty fish (92.94 ± 0.13 g) were assigned into six groups for 60 days. Control (C), ZnONPs20, and ZnONPs30 groups were fed on basal diets fortified with 0, 20, and 30 mg kg-1 ZnONPs without Cu exposure. Cu, Cu + ZnONPs20, and Cu + ZnONPs30 groups were exposed to Cu at a dose of 10 mg L-1 and fed on basal diets fortified with 0, 20, and 30 mg kg-1 ZnONPs, respectively. The results revealed that the Cu-exposed fish experienced abnormal clinical signs and behavioral changes. The growth indices and acetylcholine esterase activity were significantly decreased (P < 0.05) in the Cu group. Meanwhile, hepatorenal and serum stress indices (P < 0.05) were significantly elevated with chronic Cu exposure. In addition, a higher expression of stress (P < 0.05) (heat shock protein 60 and hypoxia-inducible factor-1 alpha) and apoptotic-related genes (C/EBP homologous protein, caspase-3, and Bcl-2 Associated X-protein) with down-regulation (P < 0.05) of the anti-apoptotic-related genes (B-cell lymphoma 2 and proliferating cell nuclear antigen) was noticed in the Cu-exposed fish. Histopathological alterations in the gills, liver, kidney, and spleen were markedly reported in the Cu-exposed group. The dietary supplementation with ZnONPs significantly alleviated the negative impacts of chronic waterborne-Cu exposure on growth performance, physiological changes, gene expression, and tissue architecture, especially at 30 mg kg-1 diet level. In particular, the inclusion of ZnONPs at the 30 mg kg-1 diet level produced better outcomes than the 20 mg kg-1 diet. Overall, ZnONPs could be added as a feed supplement in the C. gariepinus diet to boost the fish's health and productivity and alleviate the stress condition brought on by Cu exposure.

Therapeutic Role of Hemp (Cannabis sativa) Against Copper-Induced Toxicity in Labeo rohita and Cirrhinus mrigala.

For decades hemp has been used as a therapeutic agent for enhancing immunity in animals. Current study was conceptualized to find out the protective role of dietary hemp seed products (hemp seed oil (HO) and hemp seed (HS)) against copper-induced toxicity in fish. Fingerlings of Labeo rohita (Rohu) and Cirrhinus mrigala (Mrigal) were exposed to copper at 20% of the 96 h LC50 (1.34 and 1.52 ppm, respectively) for 30 days. Following Cu exposure, fish were maintained on two types of hemp (Cannabis sativa)-supplemented feeds, on graded levels of hemp seed oil (HO: 1%, 2%, 3%) and hemp seed (HS: 5%, 10%, 15%) for 50 days, while one group was the control (without any copper exposure as well as any supplementation). Copper exposure significantly increased (P < 0.05) WBCs, hematocrit, MCHV, eosinophils, and lymphocytes in L. rohita and also in C. mrigala as compared to control. Copper exposure also significantly (P < 0.05) changed lysozymes, plasma protein, and IgM in both species, in comparison to control. Moreover, alkaline phosphatase, bilirubin, serum glutamic-pyruvic transaminase, and aspartate transaminase were significantly (P < 0.05) changed by copper exposure in comparison to control in both species. Additionally, Antioxidant enzymes like catalase, superoxide dismutase, glutathione reductase, and glutathione peroxidase were also significantly (P < 0.05) increased in the brain, gills, liver, and muscle of copper-exposed group in both species as compared to control. Interestingly, all the altered parameter of blood, serum, liver function tests, and antioxidant enzymes (in different organs) because of copper toxicity were successfully reverted to normal level in hemp seed oil (HO) and hemp seed (HS)-supplemented fed groups of both species. In conclusion, hemp seed supplementation showed significant (P < 0.05) improved results against copper toxicity. Thus, it could be recommended as an animal feed ingredient for its therapeutic role.

Silicon dioxide nanoparticles suppress copper toxicity in Mentha arvensis L. by adjusting ROS homeostasis and antioxidant defense system and improving essential oil production.

Copper (Cu) is an essential micronutrient for plants; however, the excessive accumulation of Cu due to various anthropogenic activities generates progressive pollution of agricultural land and that causes a major constraint for crop production. Excess Cu (80 mg kg-1) in the soil diminished growth and biomass, photosynthetic efficiency and essential oil (EO) content in Mentha arvensis L., while amplifying the antioxidant enzyme's function and reactive oxygen species (ROS) production. Therefore, there is a pressing need to explore effective approaches to overcome Cu toxicity in M. arvensis plants. Thus, the present study unveils the potential of foliar supplementation of two distinct forms of silicon dioxide nanoparticles (SiO2 NPs) i.e., Aerosil 200F and Aerosil 300 to confer Cu stress tolerance attributes to M. arvensis. The experiment demonstrated that applied forms of SiO2 NPs (120 mg L-1), enhanced plants' growth and augmented the photosynthetic efficiency along with the activities of CA (carbonic anhydrase) and NR (nitrate reductase), however, the effects were more accentuated by Aerosil 200F application. Supplementation of SiO2 NPs also exhibited a beneficial effect on the antioxidant machinery of Cu-disturbed plants by raising the level of proline and total phenol as well as the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX) and glutathione reductase (GR), thereby lowering ROS and electrolytic leakage (EL). Interestingly, SiO2 NPs supplementation upscaled EO production in Cu-stressed plants with more pronounced effects received in the case of Aerosil 200F over Aerosil 300. We concluded that the nano form (Aerosil 200F) of SiO2 proved to be the best in improving the Cu-stress tolerance in plants.

Toxicity, physiological, and morphological alterations of Indian camphorweed (Pluchea indica) in response to excess copper.

Indian camphorweed (Pluchea indica (L.) Less.) is used as herbal tea due to the presence of volatile aromatic oils and several phytochemical compounds. The aim of this study was to assess the impact of copper (Cu) contamination on the physiology and morphology of P. indica, and the health risks associated with its consumption as tea. The cuttings of P. indica were subjected to 0 mM (control), 5 mM (low Cu), and 20 mM (excess Cu) of CuSO4 treatments for 1, 2, and 4 weeks. Thereafter, Cu contamination as well as physiological and morphological parameters were assessed. Cu accumulation was higher in the root tissues of plants (25.8 folds higher as compared to the leaves) grown under 20 mM CuSO4 for 4 weeks. This increased Cu accumulation resulted in the inhibition of root length, root fresh weight, and root dry weight. Cu concentration was found maximum (1.36 µg g-1 DW) in the leaf tissues under 20 mM Cu exposure for 4 weeks, with the highest target hazard quotient (THQ = 1.85), whereas Cu was not detected in control. Under exposure to 20 mM Cu treatment for 4 weeks, leaf greenness, maximum quantum yield of photosystem II, and photon yield of photosystem II diminished by 21.4%, 16.1%, and 22.4%, respectively, as compared to the control. Leaf temperature was increased by 2.5 °C, and the crop stress index (CSI) exceeded 0.6 when exposed to 20 mM Cu treatment for 2 and 4 weeks; however, the control had a CSI below 0.5. This led to a reduced transpiration rate and stomatal conductance. In addition, the net photosynthetic rate was also found sensitive to Cu treatment, which resulted in decreased shoot and root growth. Based on the key results, it can be suggested that P. indica herbal tea derived from the foliage of plants grown under a 5 mM Cu level (0.75 µg g-1 DW) with a target hazard quotient below one aligns with the recommended dietary intake of Cu in leafy vegetables. The study recommends choosing cuttings from plants with a small canopy as plant material in the greenhouse microclimates to validate the growth performance in the Cu-contaminated soil and simulate the natural shrub architecture and life cycle.

Oral iron supplementation ameliorated alterations in iron uptake and utilization in copper-toxic female Wistar rats.

OBJECTIVE: Women are more susceptible to both iron deficiency and copper toxicity due to monthly flow and estrogen action, respectively. Oral iron is beneficial for menstruating women and enhances erythropoiesis, but both deficiency and excess of copper impact iron absorption and mobilization. The aim of this study was to investigate the possibility of mitigating copper toxicity in female Wistar rats while supplementing with iron. METHODS: 20 female rats (160-180g) were grouped into four: Groups 1 (Control) received 0.3mls normal saline, 2- copper-toxic (100m mg/kg Copper sulphate), 3- Copper-toxic+Iron (100 mg/kg Copper sulphate + 1 mg/kg Ferrous sulphate) and 4- Iron (1 mg/kg Ferrous sulphate). All treatment was administered orally for 5 weeks. Blood was collected retro-orbitally after light anesthesia into EDTA and plain bottles for hematological, serum copper, iron, ferritin and total iron binding capacity (TIBC) analysis. Liver was excised for copper and iron levels while bone marrow was harvested for myeloid/erythroid ratio. The data were analyzed by one-Way ANOVA and statistical significance was considered at p<0.05. RESULTS: Iron supplementation significantly increased packed cell volume, hemoglobin concentration, red blood cell count and myeloid/erythroid ratio, compared to the copper-toxic group. Serum iron and TIBC were significantly increased while liver copper and iron levels reduced significantly in iron supplemented group compared to the copper-toxic group. CONCLUSIONS: Oral iron supplementation mitigated alterations in iron absorption and mobilization following copper toxicity.

Silymarin Encapsulated Liposomal Formulation: An Effective Treatment Modality against Copper Toxicity Associated Liver Dysfunction and Neurobehavioral Abnormalities in Wistar Rats.

Wilson's disease causes copper accumulation in the liver and extrahepatic organs. The available therapies aim to lower copper levels by various means. However, a potent drug that can repair the damaged liver and brain tissue is needed. Silymarin has hepatoprotective, antioxidant, and cytoprotective properties. However, poor oral bioavailability reduces its efficacy. In this study, a "thin film hydration method" was used for synthesizing silymarin-encapsulated liposome nanoparticles (SLNPs) and evaluated them against copper toxicity, associated liver dysfunction and neurobehavioral abnormalities in Wistar rats. After copper toxicity induction, serological and behavioral assays were conducted to evaluate treatment approaches. Histological examination of the diseased rats revealed severe hepatocyte necrosis and neuronal vacuolation. These cellular degenerations were mild in rats treated with SLNPs and a combination of zinc and SLNPs (ZSLNPs). SLNPs also decreased liver enzymes and enhanced rats' spatial memory significantly (p = 0.006) in the diseased rats. During forced swim tests, SLNPs treated rats exhibited a 60-s reduction in the immobility period, indicating reduced depression. ZSLNPs were significantly more effective than traditional zinc therapy in decreasing the immobility period (p = 0.0008) and reducing liver enzymes, but not in improving spatial memory. Overall, SLNPs enhanced oral silymarin administration and managed copper toxicity symptoms.

Unraveling the interaction of copper, cadmium, calcium, and nitrate on phenolics, flavonoids, and shikonin contents of Onosma dichroantha calli by statistical modeling.

Increased anthropogenic activities have led to the accumulation of certain minerals to ecotoxic levels in the environment, which could influence the secondary metabolism of plants. Shikonin, an exudate from the roots of Onosma dichroantha, is a secondary metabolite involved in plant defense and invasion success; however, the interactive effects of copper (Cu), cadmium (Cd), calcium (Ca), and nitrate (NO3) in shikonin biosynthesis and accumulation are not known. Here, the individual, curvilinear, and pairwise effects of these elements on shikonin biosynthesis in callus culture of O. dichroantha have been investigated by means of a statistical modeling approach and multivariate regression analyses. Although the main effects of the examined minerals seemed to be suppressive, their combined interactions could enhance callus growth and secondary metabolism of O. dichroantha. Accordingly, maximum values were recorded for the callus growth index (6.85 at 23.25 µM Cu, 70 mM NO3, 1 mM Ca, 27.50 µM Cd), total phenolics (24.83 mg gallic acid equivalent at 9.75 µM Cu, 70 mM NO3, 1 mM Ca, 62.50 µM Cd), total flavonoids (6.12 mg quercetin equivalent at 30 µM Cu, 80 mM NO3, 1.5 mM Ca, 45 µM Cd), and shikonin (24.33 µg g-1 FW at 9.75 µM Cu, 70 mM NO3, 2 mM Ca, 27.5 µM Cd). Overall, these data show that increasing concentrations of the examined minerals in culture medium can markedly influence the secondary metabolism of O. dichroantha cells and suggest that a comparable phenomenon may exist in a wider range of medicinal plants, grown on polluted environments, which may affect their invasive capabilities.

Immune-antioxidant trait, growth, splenic cytokines expression, apoptosis, and histopathological alterations of Oreochromis niloticus exposed to sub-lethal copper toxicity and fed thyme and/or basil essential oils enriched diets.

Two experiments were conducted in this study, using 250 Oreochromis niloticus (O. niloticus) (average weight 30.28 ± 0.27 g). The first experiment was conducted to investigate the 96-h lethal concentration 50 (LC50) of copper chloride (CuCl2) using the probit analysis, seventy fish was divided into seven different concentration of CuCl2 (0, 22, 23, 24, 25, 26, and 27 mg/L), the accurate Cu concentrations were (1.23, 5.36, 6.02, 6.98, 7.05, 7.93, 8.12 mg/L Cu). The second experiment was conducted for investigating the effect of dietary supplementation with thyme (Thymus vulgaris, T. vulgaris) and sweet basil (Ocimum basilicum, O. basilicum) essential oils (TEO and BEO respectively) against sub-lethal Cu exposure (1/10 96-h LC50 of CuCl2). About 180 fish was divided into six groups in triplicate (10 fish/replicate, 30 fish/group). Group 1 (C) was kept as a control group with no Cu exposure and was fed the control basal diet. Group 2 (C-Cu) was fed the control basal diet and simultaneously exposed to 1/10 of the 96 h LC50 of CuCl2 (2.574 mg/L) as a sub-lethal concentration of Cu, where the realistic Cu concentration was 3.976 mg/L. Group 3 (TEO) and group 4 (BEO) were fed the diets fortified with 1%TEO and BEO, respectively without exposure to Cu. Group 5 (TEO-Cu) and group 6 (BEO-Cu) were fed the diets fortified with 1%TEO and 1%BEO, respectively, and simultaneously exposed to 1/10 of the 96 h LC50 of CuCl2 (2.574 mg/L). The growth and behavioral performance, immunological response and its related gene expression, antioxidant status, stress biomarker indicators, apoptosis biomarkers, and histopathological alteration were investigated. The results of the first experiment showed that the 96-h LC50 of CuCl2 in O. niloticus was 25.740 mg/L with lower and upper confidence limits of 25.152 and 26.356 mg/L, respectively. The results of the second experiment showed that sub-lethal Cu exposure induced growth retardation (lowered final body weight, total weight gain, and specific growth rate %), behavioral abnormalities (slower swimming activity and feeding performance), immunosuppression (lowered nitric oxide, complement-3, lysozyme, total proteins, albumin, and globulin), and lowering the hepatic antioxidant functions (higher MDA, and lower SOD, CAT, and GPx) in the exposed fish. Furthermore, alteration in the immune-related genes expression (down-regulation of IL-10 and TGF-ß and up-regulation of IL-1ß, IL-6, IL-8, and TRL-4), hepato-renal dysfunction (elevated ALT, AST, urea, and creatinine), and high levels of serum stress indicators (cortisol and glucose) were markedly evident. sub-lethal Cu toxicity induced significant up-regulation of apoptosis biomarkers involving, nuclear factor-κß (NF-κß), Bcl-2 Associated X-protein (BAX), meanwhile, the expression of B-cell lymphoma 2 (BCL2) and Proliferating cell nuclear antigen (PCNA) was remarkably down-regulated. In addition, apoptosis was also evident by histopathological investigation of branchial, hepatic, and renal sections. TEO and/or BEO dietary supplementation mitigate the destructive impacts of sub-lethal Cu exposure in O. niloticus, depending on the results of our study, it could be concluded that TEO and BEO with a 1% dietary level could be a promising antioxidant, immunostimulant, anti-stress factors, and anti-apoptosis mediators against heavy metal contaminants (Cu) in O. niloticus, providing a solution to the problem of aquatic bodies pollution, consequently aiding in the development of aquaculture industry.

Boron-mediated amelioration of copper-toxicity in sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] seedlings involved reduced damage to roots and improved nutrition and water status.

'Xuegan' (Citrus sinensis) seedlings were fertilized 6 times weekly for 24 weeks with 0.5 or 350 µM CuCl2 and 2.5, 10 or 25 µM H3BO3. Cu-toxicity increased Cu uptake per plant (UPP) and Cu concentrations in leaves, stems and roots, decreased water uptake and phosphorus, nitrogen, calcium, magnesium, potassium, sulfur, boron and iron UPP, and increased the ratios of magnesium, potassium, calcium and sulfur UPP to phosphorus UPP and the ratios of leaf magnesium, potassium and calcium concentrations to leaf phosphorus concentration. Many decaying and dead fibrous roots occurred in Cu-toxic seedlings. Cu-toxicity-induced alterations of these parameters and root damage decreased with the increase of boron supply. These results demonstrated that B supplementation lowered Cu uptake and its concentrations in leaves, stems and roots and subsequently alleviated Cu-toxicity-induced damage to root growth and function, thus improving plant nutrient (decreased Cu uptake and efficient maintenance of the other nutrient homeostasis and balance) and water status. Further analysis indicated that the improved nutrition and water status contributed to the boron-mediated amelioration of Cu-toxicity-induced inhibition of seedlings, decline of leaf pigments, large reduction of leaf CO2 assimilation and impairment of leaf photosynthetic electron transport chain revealed by greatly altered chlorophyll a fluorescence (OJIP) transients, reduced maximum quantum yield of primary photochemistry (Fv/Fm), quantum yield for electron transport (ETo/ABS) and total performance index (PIabs,total), and elevated dissipated energy per reaction center (DIo/RC). To conclude, our findings corroborate the hypothesis that B-mediated amelioration of Cu-toxicity involved reduced damage to roots and improved nutrient and water status. Principal component analysis showed that Cu-toxicity-induced changes of above physiological parameters generally decreased with the increase of B supply and that B supply-induced alterations of above physiological parameters was greater in 350 µM Cu-treated than in 0.5 µM Cu-treated seedlings. B and Cu had a significant interactive influence on C. sinensis seedlings.

Mitigating the Growth, Biochemical Changes, Genotoxic and Pathological Effects of Copper Toxicity in Broiler Chickens by Supplementing Vitamins C and E.

This experiment was carried out to explore the efficiency of an individual or combined doses of vitamin C (Vit. C) and vitamin E (Vit. E) in alleviating biochemical, genotoxicity, and pathological changes in the liver induced by copper sulfate (CuSO4) toxicity in broiler chickens. Two hundred and fifty-one-day-old broiler chicks were haphazardly allotted into five groups (five replicates/group, ten chicks/replicate). The birds were fed five experimental diets; (1) basal diet with no additives (CON), (2) basal diets supplemented with 300 mg CuSO4/kg diet (CuSO4), (3) basal diets supplemented with 300 mg CuSO4/kg diet + 250 mg Vit. C /kg diet, (4) basal diets supplemented with 300 mg CuSO4/kg diet +250 mg Vit. E /kg diet, (5) basal diets supplemented with 300 mg CuSO4/kg diet + 250 mg Vit. C /kg diet + 250 mg Vit. E /kg diet for six weeks. The results displayed that CuSO4-intoxicated birds had significantly (p < 0.05) decreased bodyweight, weight gain, and feed intake with increased feed conversion ratio from the 2nd week till the 6th week compared with the CON. However, these changes were minimized by single or combined supplementation of vitamin C and E. The FCR was insignificantly different in birds-fed diets complemented with vitamin C and E singly or in combination from the 3rd week of age compared to the CON. Serum aminotransferases (ALT, AST) and alkaline phosphatase (ALP) were elevated in CuSO4-intoxicated birds (p < 0.05). Additionally, they showed a drop in serum total protein (TP), albumin, globulins, triglycerides (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), very low-density lipoprotein-cholesterol (VLDL-C), and high-density lipoprotein-cholesterol (HDL-C) levels compared to the CON (p < 0.05). Concomitantly, histopathological and DNA changes were perceived in the liver of CuSO4-intoxicated birds. Co-supplementation of Vit. C and Vit. E single-handedly or combined with CuSO4-intoxicated chickens enhanced the performance traits and abovementioned changes, especially with those given combinations of vitamins. From the extant inquiry, it could be established that supplementation of vitamin C and E was beneficial for mitigating the harmful effects of CuSO4 toxicity on growth performance and liver histoarchitecture in broiler chickens.

Alleviating Effects of Vitamins C and E Supplementation on Oxidative Stress, Hematobiochemical, and Histopathological Alterations Caused by Copper Toxicity in Broiler Chickens.

The current investigation evaluated the alleviating effects of vitamin C and vitamin E on oxidative stress, hematobiochemical, and histopathological changes in the kidney induced by copper sulfate (CuSO4) toxicity in chickens. Two hundred and fifty-one-day-old male broiler chicks were randomly allotted into five experimental groups (five replicates/group, ten chicks/replicate): 1st group-basal diet with no additives (control group), 2nd group-basal diet complemented with CuSO4 (300 mg/kg diet), 3rd group-basal diet with CuSO4 (300 mg/kg diet) + vitamin C (250 mg/kg diet), 4th group-basal diet with CuSO4 (300 mg/kg diet) + vitamin E (250 mg/kg diet), and 5th group-basal diet with CuSO4 (300 mg/kg diet) + vitamin C (250 mg/kg diet) + vitamin E (250 mg/kg diet) for a 42 day feeding period. The results showed a significant reduction in red blood cells (RBCs), hemoglobin (Hb) concentration, and hematocrit values as well as total leukocyte counts (WBCs), lymphocyte, heterophil, and monocyte counts in the CuSO4-intoxicated birds (2.42 × 106/µL, 9.54 g/dL, 26.02%, 15.80 × 103/µL, 7.86 × 103/µL, 5.26 × 103/µL, and 1.18 × 103/µL, respectively, at the 6th week) compared to (2.79 × 106/µL, 10.98 g/dL, 28.46%, 21.07 × 103/µL, 10.84 × 103/µL, 7.12 × 103/µL, and 1.60 × 103/µL, respectively) in the control group. Moreover, CuSO4-intoxicated birds showed hypoglycemia with a rise in serum uric acid and creatinine levels (122.68, 5.18, and 0.78 mg/dL at the 6th week) compared to (159.46, 4.41, and 0.61 mg/dL) in the control group. The CuSO4 toxicity in birds induced oxidative stress, indicated by a high serum malondialdehyde level (MDA) and diminished activity of the antioxidant enzymes (glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD)) (2.01 nmol/mL, 37.66 U/mL, and 2.91 U/mL, respectively, at the 6th week) compared to (1.34 nmol/mL, 57.00 U/mL, 4.99 U/mL, respectively) in the control group. High doses of Cu exposure caused severe microscopic alterations in kidney architecture. The addition of vitamins C and E, singularly or in combination, displayed a beneficial effect in alleviating these harmful effects of Cu toxicity. These findings showed the possible mitigating impacts of dietary antioxidants on the hematobiochemical alterations, oxidative stress, and kidney damage induced by CuSO4 toxicity.

Exogenous copper exposure causing clinical wilson disease in a patient with copper deficiency.

BACKGROUND: Human Swayback is a disease characterized by acquired copper deficiency which primarily manifests as myeloneuropathy. Common causes include malabsorptive disorders, gastric surgery, total parenteral nutrition and excessive zinc intake. In contrast, copper supplementation should be closely monitored as excessive doses can lead to acute intoxication and in chronic cases, cirrhosis. Copper derangements are rare, however it is important to consider them due to potential severe complications. CASE PRESENTATION: We present a middle-aged man who had been previously diagnosed with Human Swayback after presenting with various neurological symptoms. The patient was subsequently placed on copper supplementation. A decade later, he was referred to our hospital for liver transplant evaluation due to new diagnosis of decompensated end-stage liver disease after an abdominal surgery. His initial workup was suggestive of Wilson disease-subsequent ATP7B gene was negative. Ultimately, the patient underwent liver transplantation; liver explant was significant for a copper dry weight concentration of 5436 mcg/g. CONCLUSIONS: Human Swayback is a very rare copper-related disease which deserves awareness due to its potential irreversible health effects in the human body. Additionally, in patients who require copper supplementation, serial levels should be monitored to ensure adequate copper levels.

Neurotoxic Effect of Flavonol Myricetin in the Presence of Excess Copper.

Oxidative stress (OS) induced by the disturbed homeostasis of metal ions is one of the pivotal factors contributing to neurodegeneration. The aim of the present study was to investigate the effects of flavonoid myricetin on copper-induced toxicity in neuroblastoma SH-SY5Y cells. As determined by the MTT method, trypan blue exclusion assay and measurement of ATP production, myricetin heightened the toxic effects of copper and exacerbated cell death. It also increased copper-induced generation of reactive oxygen species, indicating the prooxidative nature of its action. Furthermore, myricetin provoked chromatin condensation and loss of membrane integrity without caspase-3 activation, suggesting the activation of both caspase-independent programmed cell death and necrosis. At the protein level, myricetin-induced upregulation of PARP-1 and decreased expression of Bcl-2, whereas copper-induced changes in the expression of p53, p73, Bax and NME1 were not further affected by myricetin. Inhibitors of ERK1/2 and JNK kinases, protein kinase A and L-type calcium channels exacerbated the toxic effects of myricetin, indicating the involvement of intracellular signaling pathways in cell death. We also employed atomic force microscopy (AFM) to evaluate the morphological and mechanical properties of SH-SY5Y cells at the nanoscale. Consistent with the cellular and molecular methods, this biophysical approach also revealed a myricetin-induced increase in cell surface roughness and reduced elasticity. Taken together, we demonstrated the adverse effects of myricetin, pointing out that caution is required when considering powerful antioxidants for adjuvant therapy in copper-related neurodegeneration.

Copper toxicity affects phosphorus uptake mechanisms at molecular and physiological levels in Cucumis sativus plants.

Due to the deliberate use of cupric fungicides in the last century for crop-defence programs, copper (Cu) has considerably accumulated in the soil. The concentrations of Cu often exceed the safety limits of risk assessment for Cu in soil and this may cause toxicity in plants. Copper toxicity induces nutritional imbalances in plants and constraints to plants growth. These aspects might be of paramount importance in the case of phosphorus (P), which is an essential plant macronutrient. In this work, hydroponically grown cucumber plants were used to investigate the influence of the exposure to different Cu concentrations (0.2, 5, 25 and 50 µM) on i) the phenotypic traits of plants, particularly at root level, ii) the nutrient content in both roots and shoots, and iii) the P uptake mechanisms, considering both the biochemical and molecular aspects. At high Cu concentrations (i.e. above 25 µM), the shoot and root growth resulted stunted and the P influx rate diminished. Furthermore, two P transporter genes (i.e. CsPT1.4 and CsPT1.9) were upregulated at the highest Cu concentration, albeit with different induction kinetics. Overall, these results confirm that high Cu concentrations can limit the root acquisition of P, most likely via a direct action on the uptake mechanisms (e.g. transporters). However, the alteration of root plasma membrane permeability induced by Cu toxicity might also play a pivotal role in the observed phenomenon.

Exogenous abscisic acid mediates ROS homeostasis and maintains glandular trichome to enhance artemisinin biosynthesis in Artemisia annua under copper toxicity.

One of the major abiotic stresses that cause environmental pollution is heavy metal stress. In the present investigation, copper (Cu) toxicity caused morphological and cellular damages to the Artemisia annua L. plants but supplementation of abscisic acid (ABA) ameliorated the damaging effect of Cu. Copper toxicity significantly reduced the shoot and root lengths; fresh and dry weights of shoot. However, exogenous application of ABA to Cu-treated plants significantly attenuated the damaging effects on plants caused by Cu toxicity. Copper stress also reduced the physiological and biochemical parameters, but ABA application ameliorated the negative effects of Cu in the affected plant. Accumulation of Cu in plant tissues significantly increased the membrane damage and oxidative enzyme activities such as catalase (CAT), peroxidase (POX) and superoxide dismutase (SOD). Further, the impact of high concentration of Cu on density, area and ultrastructure of glandular trichomes and artemisinin content was studied. Moreover, the foliar application of ABA improved the area, density of glandular trichomes and secured the plant cells from Cu toxicity. Therefore, this investigation indicated that the exogenous application of ABA protects A. annua plant by increasing antioxidant enzymes activity, which helps in maintaining cell integrity of leaves and results in increased artemisinin production.

Activation of HIF-1 signaling ameliorates liver steatosis in zebrafish atp7b deficiency (Wilson's disease) models.

Wilson's disease is an autosomal recessive disease characterized by excess copper accumulated in the liver and brain. It is caused by mutations in the copper transporter gene ATP7B. However, based on the poor understanding of the transcriptional program involved in the pathogenesis of Wilson's disease and the lack of more safe and efficient therapies, the identification of novel pathways and the establishment of complementary model systems of Wilson's disease are urgently needed. Herein, we generated two zebrafish atp7b-mutant lines using the CRISPR/Cas9 editing system, and the mutants developed hepatic and behavioral deficits similar to those observed in humans with Wilson's disease. Interestingly, we found that atp7b-deficient zebrafish embryos developed liver steatosis under low-dose Cu exposure, and behavioral deficits appeared under high-dose Cu exposure. Analyses of publicly available transcriptomic data from ATP7B-knockout HepG2 cells demonstrated that the HIF-1 signaling pathway is downregulated in ATP7B-knockout HepG2 cells compared with wildtype cells following Cu exposure. The HIF-1 signaling pathway was also downregulated in our atp7b-deficient zebrafish mutants following Cu exposure. Furthermore, we demonstrate that activation of the HIF-1 signaling pathway with the chemical compound FG-4592 or DMOG ameliorates liver steatosis and reduces accumulated Cu levels in zebrafish atp7b deficiency models. These findings introduce a novel prospect that modulation of the HIF-1 signaling pathway should be explored as a novel strategy to reduce copper toxicity in Wilson's disease patients.

Human Embryonic Stem Cell-Derived Wilson's Disease Model for Screening Drug Efficacy.

Human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) have been extensively studied as an alternative cellular model for recapitulating phenotypic and pathophysiologic characters of human diseases. Particularly, hiPSCs generated from the genetic disease somatic cells could provide a good cellular model to screen potential drugs for treating human genetic disorders. However, the patient-derived cellular model has a limitation when the patient samples bearing genetic mutations are difficult to obtain due to their rarity. Thus, in this study, we explored the potential use of hPSC-derived Wilson's disease model generated without a patient sample to provide an alternative approach for modeling human genetic disease by applying gene editing technology. Wilson's disease hPSCs were generated by introducing a R778L mutation in the ATP7B gene (c.2333G>T) using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system into wildtype hESCs. Established Wilson's disease hESCs were further differentiated into hepatocyte-like cells (HLCs) and analyzed for disease phenotypes and responses against therapeutic agent treatment. R778L mutation in the ATP7B gene was successfully introduced into wildtype hESCs, and the introduction of the mutation neither altered the self-renewal ability of hESCs nor the differentiation capability into HLCs. However, R778L mutation-introduced HLCs exhibited higher vulnerability against excessive copper supplementation than wildtype HLCs. Finally, the applicability of the R778L mutation introduced HLCs in drug screening was further demonstrated using therapeutic agents against the Wilson's diseases. Therefore, the established model in this study could effectively mimic the Wilson's disease without patient's somatic cells and could provide a reliable alternative model for studying and drug screening of Wilson's disease.

Bee glue (propolis) improves reproductive organs, sperm quality and histological changes and antioxidant parameters of testis tissues in rats exposed to excess copper.

This study was designed to determine the effects of propolis on the sperm quality, antioxidant and histological parameters in the testicular tissues of male Sprague Dawley rats exposed to excessive copper (Cu). In this aim, 24 rats were randomly divided into four groups as follows: the control, Cu, Propolis and Cu+Propolis. When compared to control group, Cu administration significantly decreased sperm motility and concentration, increased total abnormal sperm rate. It caused a significant induction the malondialdehyde (MDA), and reduction the superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) in testicular tissues. Also, it caused loss, disorganisation and vacuolation of the germinal epithelium, oedema of the interstitial tissues, proliferation of the interstitial cells, spilled immature spermatogenic cells in the lumen of some seminiferous tubules. A large number of active caspase-3-positive stained apoptotic cells and a significant decrease in Johnsen's testicular score were determined. However, significant ameliorations were observed in all sperm characteristics, MDA, SOD, CAT, GSH, seminiferous tubules, number of apoptotic cells and Johnsen's testicular score in Cu+Propolis group. Our results showed that oral supplementation of propolis had curative effect on the sperm quality, antioxidant and histological parameters in the testicular tissues of male Sprague Dawley rats exposed to Cu.

Melatonin: A hypothesis regarding its use to treat Wilson disease.

Wilson disease is associated with excessive copper accumulation in cells, primarily in the liver and brain. The subcellular lesions caused by an excess of this essential metal accounts for many of the signs of Wilson disease. The drugs used to treat this disease are not always effective, and depending on dose, they may have collateral toxicity. Melatonin is an endogenously-produced molecule that functions as a copper chelator, a potent antioxidant, and as a suppressor of endoplasmic reticulum stress and the unfolded protein response in both the liver and the brain, while also reducing fibrosis/cirrhosis in the liver. Melatonin is inexpensive, non-toxic and can be administered via any route. Melatonin should be tested for its potential utility in experimental models of Wilson disease with extension to the human if melatonin proves to be effective in the animal studies.