Dysregulation of intracellular copper trafficking pathway in a mouse model of mutant copper/zinc superoxide dismutase-linked familial amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are responsible for 20% of familial amyotrophic lateral sclerosis through a gain-of-toxic function. We have recently shown that ammonium tetrathiomolybdate, an intracellular copper-chelating reagent, has an excellent therapeutic benefit in a mouse model for amyotrophic lateral sclerosis. This finding suggests that mutant SOD1 might disrupt intracellular copper homeostasis. In this study, we investigated the effects of mutant SOD1 on the components of the copper trafficking pathway, which regulate intracellular copper homeostasis. We found that mutant, but not wild-type, SOD1 shifts intracellular copper homeostasis toward copper accumulation in the spinal cord during disease progression: copper influx increases, copper chaperones are up-regulated, and copper efflux decreases. This dysregulation was observed within spinal motor neurons and was proportionally associated with an age-dependent increase in spinal copper ion levels. We also found that a subset of the copper trafficking pathway constituents co-aggregated with mutant SOD1. These results indicate that the nature of mutant SOD1 toxicity might involve the dysregulation of the copper trafficking pathway, resulting in the disruption of intracellular copper homeostasis.     

A proteomic investigation into the human cervical cancer cell line HeLa treated with dicitratoytterbium (III) complex

Lanthanides have been reported to induce apoptosis in cancer cell lines. Human cervical cancer cell line HeLa was found to be more sensitive to dicitratolanthanum (III) complex ([LaCit₂]³⁻) than other cancer cell lines. However, the effect and mechanism of dicitratoytterbium (III) complex ([YbCit₂]³⁻) on HeLa cells is unknown. Using biochemical and comparative proteomic analyses, [YbCit₂]³⁻ was found to inhibit HeLa cell growth and induce apoptosis. Similar to the effects of [LaCit₂]³⁻, proteomics results from [YbCit₂]³⁻-treated cells revealed profound changes in proteins relating to mitochondria and oxidative stress, suggesting that mitochondrial dysfunction plays a key role in [YbCit₂]³⁻-induced apoptosis. This was confirmed by the decreased mitochondrial transmembrane potential and the increased generation of reactive oxygen species in [YbCit₂]³⁻-treated cells. Western blot analysis showed that [YbCit₂]³⁻-induced apoptosis was accompanied by the activation of caspase-9 and specific proteolytic cleavage of PARP, leading to an increase in the pro-apoptotic protein Bax and a decrease in the anti-apoptotic protein Bcl-2. These results suggest a mitochondrial pathway of cell apoptosis in [YbCit₂]³⁻-treated cells, which will help us understand the molecular mechanisms of lanthanide-induced apoptosis in tumor cells.     

Relevance of Non-ceruloplasmin Copper to Oxidative Stress in Patients with Hepatocellular Carcinoma

Altered copper homeostasis and oxidative stress have been observed in patients with hepatocellular carcinoma. Non-ceruloplasmin copper, the free form, is a potent pro-oxidant than the protein bound copper. The aim of the present study was to evaluate which form of copper can be correlated with the oxidative stress in the circulation and in the malignant liver tissues of hepatocellular carcinoma patients. Hepatocellular carcinoma patients (grades II and III, n = 18) were enrolled in this study. Serum levels of total, free and bound copper, ceruloplasmin, iron, iron-binding capacity, lipid peroxidation products, and enzymatic and non-enzymatic antioxidants were quantified in serum and in malignant liver tissues and compared with those of normal samples (n = 20). A significant positive correlation between the serum non-ceruloplasmin copper and lipid peroxidation products and negative correlation with antioxidants were observed in hepatocellular carcinoma patients. In liver tissue, glutathione peroxidase, superoxide dismutase, and catalase activity were significantly decreased with concomitant elevation in oxidative stress markers. Our experiment revealed that the elevation in non-ceruloplasmin copper has high relevance with the oxidative stress than the bound copper.     

Accumulation of copper and zinc from liquid manure in agricultural soils and crop plants

In Northern Italy it is a common practice to utilise slurries and other manure from intensive animal farming to fertilise agricultural soils. Due to the relatively high copper and zinc contents of these materials, this practice could lead to contamination of soils and the crops grown on them. In this study we have evaluated the extent of copper and zinc contamination of soils subjected to different levels of fertilisation with liquid manure and the copper and zinc concentrations in the edible tissues of three crops (maize, sugar beet and lucerne) grown on the same soils. There was a direct correlation between soil copper and zinc contents and the levels of application of animal wastes and copper concentrations were high enough to represent a risk, according to current European legislation. The metal contents in the edible tissues of the three crops were relatively low and variable, but showed no clear correlations with the intensity of liquid manure applications. Overall, there was no risk of contamination of the food chain, as all concentrations were well below levels considered to be toxic to animals.     

Chromium,Copper, and Arsenic Concentrations in Soil Underneath CCA-Treated Wood Structures

Soils below nine structures (decks and foot bridges) in Florida were examined to evaluate potential impacts from chromated copper arsenate (CCA), a common wood preservative. Eight of the nine structures were confirmed to have been treated with CCA. Soils collected were evaluated for arsenic, chromium, and copper concentrations as well as pH, volatile solids content and particle size distribution. Two types of soil samples were collected: a soil core and surface soil samples (upper 2.5 cm). One soil core was collected from below each deck and one control core was collected from an area removed from one of the structures. Eight or nine surface soil samples were collected in a grid-like fashion from beneath each structure. Equal numbers of surface control samples were collected from areas away from the structures. Metal concentrations were elevated in both the soil cores and surface samples collected from below the CCA-treated structures. Core samples showed elevated concentrations of metals at depths up to 20 cm. The arithmetic mean concentrations of arsenic, chromium, and copper in the 65 surface soil samples collected from below CCA-treated structures were 28.5 mg/kg, 31.1 mg/kg, and 37.2 mg/kg, respectively, whereas the mean concentrations of arsenic, chromium, and copper in the control samples were 1.34 mg/kg, 8.62 mg/kg, and 6.05 mg/kg, respectively. Arsenic concentrations exceeded Florida's risk-based soil cleanup target level (SCTL) for residential settings in all 65 surface soil samples. The industrial setting SCTL was exceeded in 62 of the 65 samples.     

Effects of Cadmium and Mixed Heavy Metals on Rice Growth in Liaoning,China

Joint effects of Cd and other heavy metals (Pb, Cu, Zn and As) on the growth and development of rice plants and the uptake of these heavy metals by rice were studied using the pot-culture method combined with chemical and statistical analyses. The results showed that the growth and development of rice plants were strongly influenced by the double-element combined pollution. There was an average decrease in the height of rice plants of 4.0–5.0 cm, and grain yield was decreased by 20.0–30.0%, compared with the control. The uptake of Cd by rice plants was promoted due to the interactions between Cd and the other heavy metals added to the soil. The Cd concentration in roots, stems/leaves and seeds increased 31.6–47.7, 16.7–61.5 and 19.6–78.6%, respectively. Due to interactions, uptake of Pb, Cu and Zn by roots and stems/leaves was inhibited, accumulation of Pb, Cu and Zn in seeds was increased, uptake of As by roots was promoted and uptake of As by stems/leaves was inhibited. In particular, the upward transporting ability of the heavy metals absorbed by rice plants was significantly increased.     

Copper kinetics and hepatic metallothionein levels in the frog Rana ridibunda, after exposure to CuCl2

Adult female frogs Rana ridibunda were exposed to 50 and 100 ppm of Cu (as CuCl₂) dissolved in water for 5, 15 and 30 days. We measured the Cu content in the liver, kidneys, ventral skin, and large intestine. Hepatic metallothionein (MT) was also measured and we identified by elution the type of proteins bound to copper. Gross morphological characteristics of the frogs were not affected by Cu accumulation. Cu uptake took place first across the skin, then accumulated first in the large intestine, and then in the liver which was continuously accumulating Cu at all exposure concentrations and times. The highest concentration of the metal was recorded in the kidneys at 30 days and 100 ppm exposure. It appears that the kidneys act as the secondary route of Cu detoxification, probably after a Cu overload of liver. The concentration of hepatic MT increased with the increase of Cu concentration in liver at the 5^th and 15^th day of exposure but we observed a decrease by the end of the experiment. Cu was observed in the MT-fraction, and in the high-molecular weight protein fraction.     

Double base lesions of DNA by a metabolite of carcinogenic benzo[a]pyrene.

Carcinogenic benzo[a]pyrene (BP) is generally considered to show genotoxicity by forming DNA adducts of its metabolite, BP-7,8-diol-9,10-epoxide. We investigated oxidative DNA damage and its sequence specificity induced by BP-7,8-dione, another metabolite of BP, using (32)P-5'-end-labeled DNA. Formamidopyrimidine-DNA glycosylase treatment induced cleavage sites mainly at G residues of 5'-TG-3' sequence and at poly(C) sequences, in DNA incubated with BP-7,8-dione in the presence of NADH and Cu(II), whereas piperidine treatment induced cleavage sites at T mainly of 5'-TG-3'. BP-7,8-dione strongly damaged the G and C of the ACG sequence complementary to codon 273 of the p53 gene. Catalase and a Cu(I)-specific chelator attenuated the DNA damage, indicating the involvement of H(2)O(2) and Cu(I). BP-7,8-dione with NADH and Cu(II) also increased 8-oxo-7,8-dihydro-2'-deoxyguanosine formation. We conclude that oxidative DNA damage, especially double base lesions, may participate in the expression of carcinogenicity of BP in addition to DNA adduct formation.     

Does the tube of a benthic chironomid larva play a role in protecting its dweller against chemical toxicants?

A laboratory culture of an Israeli benthic midge, Chironomus luridus, was exposed to two chemicals: copper and monochloramine. The objective of this study was to determine the protective nature of Chironomus' larval tube. Three experimental conditions were tested: larva with sand tubes, with silt tubes and without tubes. Larvae without tubes were significantly more sensitive to copper and chloramine than larvae that had sand or silt as tube building substrate. The tubes protected the insects against chemicals throughout 14 days of exposure time. Silt tubes had higher protective value than sand tubes, especially when exposed to copper for a short period of time (LC₅₀/ 24 h, with silt, sand, or none: 80.0, 7.0 and 3.4 mg l^–1 copper, respectively). C. luridus seemed to be better protected against copper than against chloramine (LC₅₀/ 24 h, with silt, sand, or none: 12.2, 6.4 and 3.7 mg l^–1 chloramine, respectively). The acute toxicity of copper to chironomid larvae was investigated using a cytochemical method. Larva in silt tubes had significantly higher non-specific esterase activity in midgut cells than larvae without tubes. We conclude that, in addition to its role in feeding, respiration and anti-predation shelter, the C. luridus tube protects its inhabitant from toxic substances.