Bathocuproinedisulfonic Acid Disodium Salt Research Articles

A novel method of rapid detection for heavy metal copper ion via a specific copper chelator bathocuproinedisulfonic acid disodium salt

The extensive usage and production of copper may lead to toxic effects in organisms due to its accumulation in the environment. Traditional methods for copper detection are time consuming and infeasible for field usage. It is necessary to discover a real-time, rapid and economical method for detecting copper to ensure human health and environmental safety. Here we developed a colorimetric paper strip method and optimized spectrum method for rapid detection of copper ion based on the specific copper chelator bathocuproinedisulfonic acid disodium salt (BCS). Both biological assays and chemical methods verified the specificity of BCS for copper. The optimized reaction conditions were 50 mM Tris–HCl pH 7.4, 200 µM BCS, 1 mM ascorbate and less than 50 µM copper. The detection limit of the copper paper strip test was 0.5 mg/L by direct visual observation and the detection time was less than 1 min. The detection results of grape, peach, apple, spinach and cabbage by the optimized spectrum method were 0.91 μg/g, 0.87 μg/g, 0.19 μg/g, 1.37 μg/g and 0.39 μg/g, respectively. The paper strip assays showed that the copper contents of grape, peach, apple, spinach and cabbage were 0.8 mg/L, 0.9 mg/L, 0.2 mg/L, 1.3 mg/L and 0.5 mg/L, respectively. These results correlated well with those determined by inductively coupled plasma-mass spectrometry (ICP-MS). The visual detection limit of the paper strip based on Cu-BCS-AgNPs was 0.06 mg/L. Our study demonstrates the potential for on-site, rapid and cost-effective copper monitoring of foods and the environment.

Oxidative stress and apoptotic effects of copper and cadmium in the zebrafish liver cell line ZFL

Copper (Cu) and cadmium (Cd) are widely used in industrial activities, resulting in Cu and Cd contamination in aquatic systems worldwide. Although Cu plays an essential role in many biological functions, an excessive amount of the metal causes cytotoxicity. In contrast, Cd is a non-essential metal that usually co-exists with Cu. Together, they cause oxidative stress in cells, leading to cell damage. These metal ions are also believed to cause cell apoptosis. In this study, we used a zebrafish liver cell line, ZFL, to study combined Cu and Cd cytotoxicity. Although Cd is more toxic than Cu, both were found to regulate the expression of oxidative stress related genes, and neither significantly altered the activity of oxidative stress related enzymes. Co-exposure tests with the antioxidant N-acetyl-l-cysteine and the Cu chelator bathocuproinedisulfonic acid disodium salt demonstrated that Cd toxicity was due to the oxidative stress caused by Cu, and that Cu at a low concentration could in fact exert an antioxidant effect against the oxidative stress in ZFL. Excessive Cu concentration triggered the expression of initiator caspases (caspase 8 and caspase 9) but suppressed that of an executioner caspase (caspase 3), halting apoptosis. Cd could only trigger the expression of initiator caspases; it could not halt apoptosis. However, a low concentration of Cu reduced the mitochondrial superoxide level, suppressing the Cd-induced apoptotic effects in ZFL.

Accumulation and Analysis of Cuprous Ions in a Copper Sulfate Plating Solution.

Knowledge of the behavior of cuprous ions (monovalent copper ion: Cu(I)) in a copper sulfate plating bath is important for improving the plating process. We successfully developed a method to quantitatively and easily measure Cu(I) in a plating solution and used it for evaluation of the solution. In this paper, a quantitative absorption spectrum measurement and a time-resolved injection measurement of Cu(I) concentrations by a color reaction are described. This procedure is effective as a method to reproduce and elucidate the phenomenon occurring in the plating bath in the laboratory. First, the formation and accumulation process of Cu(I) in solution by electrolysis of a plating solution is shown. The amount of Cu(I) in the solution is increased by electrolysis at higher current values ​​than the usual plating process. For the determination of Cu(I), BCS (bathocuproinedisulfonic acid, disodium salt), a reagent that selectively reacts with Cu(I), is used. The concentration of Cu(I) can be calculated from the absorbance of the Cu(I)-BCS complex. Next, the time measurement of the color reaction is described. The color reaction curve of Cu(I) and BCS measured by the injection method can be decomposed into an instantaneous component and a delay component. By analysis of these components, the holding structure of Cu(I) can be clarified, and this information is important when predicting the quality of the plating film to be produced. This method is used to facilitate the evaluation of the plating bath in the production line.

Quenching characteristics of bathocuproinedisulfonic acid, disodium salt in aqueous solution and copper sulfate plating solution

Bathocuproinedisulfonic acid, disodium salt (BCS) is generally used to detect Cu(I) through a color reaction. We newly found BCS fluorescence in the visible blue region in an aqueous solution. However, the fluorescence mechanism of BCS is not well known, so we should investigate its fundamental information. We confirmed that the characteristics of fluorescence are highly dependent on the molecular concentration and solvent properties. In particular, owing to the presence of the copper compound, the fluorescence intensity extremely decreases. By fluorescence quenching, we observed that a copper compound concentration of 10−6 mol/L or less could easily be measured in an aqueous solution. We also observed BCS fluorescence in copper sulfate plating solution and the possibility of detecting monovalent copper by fluorescence reabsorption.

Cu-T1 Sensor for Versatile Analysis.

Conventional magnetic sensors usually employ Fe-based magnetic materials as signal probes. In this work, we find that Cu(II) is also a useful longitudinal relaxation time (T1) signal-based magnetic probe. We adopt bathocuproinedisulfonic acid disodium salt hydrate (BCS) to chelate Cu(I) and form a stable Cu(I)-BCS complex in aqueous solution and find the significant difference in the T1 value of water protons between Cu(II) aqueous solution and Cu(I)-BCS complex aqueous solution. Redox reaction can convert Cu(II) to Cu(I) followed by the complexation of BCS, which results in apparent change of T1 that can serve as magnetic signal readout, which is the basis of this Cu-T1 sensor. Many redox reactions between Cu(II) and Cu(I) allow this Cu-T1 sensor to not only realize "one-step mode" assay such as ascorbic acid, protein, and alkaline phosphatase but also enable "multi-step mode" immunoassay, such as biomacromolecules and small molecules. This Cu-T1 sensor employs Cu ion as signal readout, providing an alternative tool for biochemical analysis.

Spectroscopic and Electrochemical Analysis of Cu(I) Complex of Copper Sulfate Electroplating Solution and Evaluation of Plated Films

In this study, we investigated a behavior of cuprous ion (denoted as Cu(I)) in the copper sulfate plating solution and effects of the Cu(I) on a quality of Cu plating film. Through spectroscopic method, two kinds of holding structure of Cu(I) in the plating solution were firstly indicated by analyzing the kinetics of complex forming reaction between the Cu (I) and bathocuproinedisulfonic acid, disodium salt (BCS). One component is ignited from Cu(I) probably incorporated into deep inside of polyethylene glycol (PEG), and the Cu(I) must have steric hindrance of PEG to be interacted with the BCS in plating solution. Another one is a component for Cu(I) which can be instantaneously reacts to the BCS to be complex. In addition, interestingly, it has been shown that instantaneous component is affecting the roughness of Cu plating film. These results indicate that spectroscopic measurement and analysis is effective for the management and evaluation of the plating solution for high quality production.

Spectroscopic and Electrochemical Analysis of Cu(I) Complex of Copper Sulfate Electroplating Solution and Evaluation of Plated Films

With density growth of printed circuit board and the development of TSV plating technology, it becomes more important to management of the plating solution during the manufacturing process. In copper sulfate electroplating, accurate measurement of the monovalent copper ions (Cu(I)) concentrations in the solutions is required, because they are pointed out to cause roughness of the plated surface. We have succeeded in analyzing the Cu(I) contained in the plating solutions conducted by absorption of a chelate of Cu(I) with bathocuproinedisulfonic acid, disodium salt (BCS). Although Cu(I) in the solutions is considered to not be detected because they are oxidized immediately, in fact, a large amount of Cu(I) is contained in the plating solution of the production lines. However, there is not enough conclusions about the effect of Cu(I) in the solution is on the plated surface. We had found that formation and accumulating Cu (I) by electrolysis under suitable conditions for copper sulfate plating solutions. Here, we will discuss a mechanism of Cu(I) accumulation in CuSO4 plating solution during the electrolysis from the view point of (1) dynamics on formation of 1:2 complex between Cu(I) and BCS chelate and (2) MALDI-MS analysis of the plating solution. In addition, by considering of implicit knowledge about the role of Cu(I) for product quality in production line, we will discuss (3) the relationship of Cu(I) amount and qualities of the Cu plated surface. (1) Dynamics on formation of 1:2 complex between Cu(I) and BCS chelate Cu(I) concentration in the plating solution can be determined from the absorbance of Cu (I) and BCS chelate. Furthermore, it is possible to classify the holding structures of Cu(I) by analyzing the kinetics reaction between BCS and Cu (I). Reaction of BCS with Cu (I) can be divided into three components, the instantaneous component of the time constant is less than 1 seconds (A0), an fast component is completed within a few seconds (AS), and components having a long time constant which is slow than a few minutes (AL). Although Cu(I) concentration in the solution was increased by electrolysis, change in each of the reaction components was not uniform (AS component was not observed in this experiment). That AL component is increased in the initial (<4min) of the electrolysis, A0 component was increased with a delay to between 6 and 10 minutes. This suggests that some Cu(I) holding structures occurring at different processes in solution. (2) MALDI-MS analysis of Cu plating solution Results of MALDI-MS analysis show some intermolecular interaction between Cu(I) and BCS. It can be considered that the some of the Cu(I) ion must be incorporated into poly ethylene glycoside (PEG) and stabilized for accumulation in the Cu plating solution. In the case of deeply incorporating into the PEG chain, Cu(I) must be isolated from the solution and attack from BCS by the PEG, and formation of Cu(I)-BCS2 complex was probably inhibited to be long kinetics component (AL). On the other hand, A0 component is not be described in clear, these Cu (I) is considered to be held in a state to quickly react with BCS. (3) Relationship of Cu(I) amount and qualities of the Cu plated surface Cu(I) in these states would have played a different role each in the electroplating process. So far, the results of the plating solution analyzing of the production line, mainly A0 component is suggested to affect the roughness of the plated surface. This effect is also supported by electrochemical measurements. Changes in the A0 values for the current time are correlated well with the changes in the area values in CVS (Cyclic Voltammetry Stripping) measurement. On the other hand, no clear relationship was observed between the AL values and area values. PEG might have to reduce the impact on the film surface by incorporating excess Cu(I). In order to investigate the effect of Cu(I) to the structures of the plating, surface were electroplated onto the copper plates with electrolysis solutions (electrolysis time : 0 to 20 minutes). In the film formed in 4 min electrolysis solution (negligible A0), roughness on the plate surface did not occur. However, in the 6 to 10 min electrolysis solutions, roughness occurs on the film surface depending on the electrolysis time. It was confirmed that Cu(I) accumulation (especially A0 component) is affecting the defect occurrence of coating appearance. These results show that spectroscopic methods for the analysis of Cu(I) in solution are effective, and the performance change of the solutions can be easily evaluated.

Control of Accumulation of Cu(I) in Copper Sulfate Electroplating Plating Solution

With density growth of printed circuit board and the development of through-silicon via (TSV) plating technology, it becomes more important to fine management of the plating solution during the manufacturing process in order to keep high quality of product. In copper sulfate electroplating, monovalent copper ions (cuprous ion: Cu(I)) has been implicitly pointed out as one of the main cause of low quality such as high roughness and dull metallic on the Cu plated surface, therefore accurate measurement of Cu(I) concentrations has been strongly required in the plating process. In spite of the needs in industry, it has been remaining as challenge to measure and monitor the Cu(I) concentration in the process because of the unstableness of Cu(I) in solution, conceitedly rapid oxidation. In fact, although a large amount of Cu(I) can be considered to be contained in the plating solution in the production lines, there is not enough knowledge about the holding structure of Cu(I) in the plating solutions. We have succeeded in optical analyzing the Cu(I) containing in the plating solutions conducted by absorption of a chelate of Cu(I) with bathocuproinedisulfonic acid, disodium salt (BCS). Here, we will report the formation and accumulation of Cu(I) in copper sulfate electroplating solutions and discuss the relationship Cu(I) activity to electrolysis conditions such as electro density, time and dissolving gas, based on the experiments in the laboratory. These results also will be important knowledge to control a process factor such as concentrations and holding structures of Cu(I) in the plating solutions even in manufactural plating line. As experimental, a time course of Cu(I) concentrations were determined from change in absorbance originated from formation of Cu(I) and BCS chelate accompanied by applying electrolysis the copper sulfate plating solutions. The electrolysis was carried out using two electrode system; anode: copper plate, cathode: platinum plate (electrochemically active area: 20×40 mm). The plating solution (150mL) was degassed with nitrogen gas through the energization. Cu(I) does not include most in the new liquid of copper sulfate, however about 1mM Cu(I) was accumulated by electrolysis for 10 minutes at a current density of 62.5mA / cm2. Cu(I) concentration in the solutions were firstly increased depending on the electrolysis time, the decreases as longer than a certain period of time, for example, about 15 minutes in 62.5mA/ cm2. Accumulation of Cu(I) tends to increases in accordance with the current density but there was no change in the following 15.5 mA/ cm2. On the contrary, the amount of Cu(I) to be accumulated even more than 78.1mA/ cm2 was less than the amount of 62.5mA/ cm2. Accumulation of Cu(I) is considered to be proportional to the current density and time within a certain range. For optimal current value in the formation of the plating film is less than this lower limit, usually the accumulation of monovalent copper would be less likely to occur. On the other hand, if it exceeds the upper limit, the precipitation of a large amount of particles in the solution was observed. In previous discussion, Cu(I) that is formed in a solution concluded that are stabilized incorporated into PEG. Excessive Cu(I) might form the copper particles by a disproportionation reaction. Through the optical analysis of Cu(I), it found that the formation and accumulation of Cu(I) depends on other conditions in electrolysis. For example, the accumulation of Cu(I) is hardly observed using phosphorus-containing copper plate as the anode instead of Cu plate. Further, when bubbling with air instead of nitrogen gas, accumulation of Cu (I) was reduced to approximately half. It can be explained that Cu (I) was oxidized by dissolved oxygen come from the bubbled air. The oxidation is a major factor that inhibits the accumulation of Cu (I). In fact by the air bubbling, Cu (I) concentration in the solution is reduced in accordance with the bubbling time. Cu (I) is more stably maintained in the degassed solution. As conclusion, these results clarified that accumulation of Cu (I) of the copper sulfate plating solution is governed by various factors, such as current-density, operation time electrode and kind of dissolved gas in electrolysis. It also shows the possibility of controlling the density and the holding structure of Cu (I) in solution. The Cu (I) is a cause of roughness of the film, it is also considered to have a positive effect on the filling in TSV plating. By controlling the formation and accumulation of Cu (I) of the copper sulfate plating solution, thereby enabling more detailed consideration of these issues.

Validation of an automated assay for the measurement of cupric reducing antioxidant capacity in serum of dogs.

BackgroundThe objective of the present study was to optimize and validate an automated method to assess the total antioxidant capacity (TAC) in serum of dogs using the cupric reducing antioxidant capacity (CUPRAC) methodology (TACc) with bathocuproinedisulfonic acid disodium salt as chelating agent, evaluating also possible variations due to the use of two different automated analyzers. The method is based on the reduction of Cu2+ into Cu1+ by the action of the non-enzymatic antioxidants that are present in the sample.ResultsImprecision was low in both apparatus utilized, and the results were linear across serial Trolox and canine serum samples dilutions. Lipids did not interfere with the assay; however, hemolysis increased the TACc concentrations. When TACc concentrations were determined in ten healthy (control) dogs and in twelve dogs with inflammatory bowel disease (IBD), dogs with IBD had lower TACc concentrations when compared with the healthy dogs.ConclusionsThe method validated in this paper is precise, simple, and fast and can be easily adapted to automated analyzers.

Endogenous ergothioneine is required for wild type levels of conidiogenesis and conidial survival but does not protect against 254 nm UV-induced mutagenesis or kill

Ergothioneine, a histidine derivative, is concentrated in conidia of ascomycetous fungi. To investigate the function of ergothioneine, we crossed the wild type Neurospora crassa (Egt+) and an ergothioneine non-producer (Egt−, Δegt-1, a knockout in NCU04343.5) and used the Egt+ and Egt− progeny strains for phenotypic analyses. Compared to the Egt+ strains, Egt− strains had a 59% reduction in the number of conidia produced on Vogel’s agar. After storage of Egt+ and Egt− conidia at 97% and 52% relative humidity (RH) for a time course to either 17 or 98days, respectively, Egt− strains had a 23% and a 18% reduction in life expectancy at 97% and 52% RH, respectively, compared to the Egt+ strains. Based on a Cu(II) reduction assay with the chelator bathocuproinedisulfonic acid disodium salt, ergothioneine accounts for 38% and 33% of water-soluble antioxidant capacity in N. crassa conidia from seven and 20day-old cultures, respectively. In contrast, ergothioneine did not account for significant (α=0.05) anti-oxidant capacity in mycelia, which have lower concentrations of ergothioneine than conidia. The data are consistent with the hypothesis that ergothioneine has an antioxidant function in vivo. In contrast, experiments on the spontaneous mutation rate in Egt+ and Egt− strains and on the effects of 254nm UV light on mutation rate and conidial viability do not support the hypothesis that ergothioneine protects DNA in vivo.

Toxicity of low doses of ultrafine diesel exhaust particles on bovine brain microvessel endothelial cells

Diesel exhaust particles (DEPs) are an important risk factor for various health conditions including neurodegenerative diseases. However, information regarding the toxicity of low doses of ultrafine DEPs in relation to the brain is limited. This study was performed to evaluate the neurotoxicity of low doses of ultrafine DEPs (ng/mL level) using bovine brain microvessel endothelial cells (BMECs) as a model of the blood-brain barrier (BBB). The cells were exposed to various concentrations of DEPs (from 1.28 ng/mL to 20 μg/mL) for 24 h, and cytotoxicity was evaluated by assaying lactate dehydrogenase activity, the generation of reactive oxygen species (ROS) by the dichlorofluorescein diacetate method, total antioxidant capacity (TAC) using bathocuproinedisulfonic acid disodium salt, and cell permeability by measuring the flux of fluorescein. The results showed that low doses of DEPs decreased cell viability and induced oxidative stress by increasing ROS generation and decreasing TAC in a dose-dependent manner. Furthermore, DEPs exposure increased BMECs permeability. This study shows that exposure to low doses of ultrafine DEPs might disrupt the function of BMECs and generate oxidative stress, suggesting that exposure to DEPs even at extremely low doses may compromise the integrity of the BBB and induce adverse effects in the central nervous system.

Analysis of Cu(I) Complexes in Copper Sulfate Electroplating Solution by Using Reaction Kinetics with a Chelate Reagent

Analysis of Cu(I) in copper sulfate electroplating solutions was conducted by absorption of a chelate of Cu(I) with bathocuproinedisulfonic acid, disodium salt (BCS). The absorbance of the color reaction of Cu(I) increased quickly in a few minutes after mixing with the chelate reagent and subsequently continued to increase slowly. To analyze the reaction kinetics of the color reaction, we divided Cu(I) complexes into two groups, small complexes and large Cu(I)-PEG complexes, and assumed the reaction of each group of complexes and BCS is a first order reaction with a specific reaction rate constant. We simulated the absorption curve with a good correlation and obtained the concentrations and the time constants, inverse of rate constants, of small complexes and large Cu(I)-PEG complexes. These concentrations and time constants are important parameters to control plating solutions. The time constant of small complexes can be attributed to the variation of the proportions of these small complexes. The smaller time constant of Cu(I)-PEG complexes can be considered the larger size distribution of Cu(I)-PEG with different chain lengths. Using this analysis, we monitored the variation of Cu(I) concentration in production lines for one month and found the increase of small complexes during the resting state of the lines. This increase corresponded to the tendency of occurring of brightening troubles.

Novel method for rapid copper chelation assessment confirmed low affinity of D-penicillamine for copper in comparison with trientine and 8-hydroxyquinolines

Copper is an essential trace element involved in many physiological processes. Since disorder of copper homeostasis is observed in various pathologies, copper chelators may represent a promising therapeutic tool. This study was aimed at: 1) formation of an in vitro methodology for screening of copper chelators, and 2) detailed analysis of the interaction of copper with clinically used D-penicillamine (D-PEN), triethylenetetramine (trientine), experimentally tested 8-hydroxyquinolines, and the disodium salt of EDTA as a standard chelator. Methodology based on bathocuproinedisulfonic acid disodium salt (BCS), usable at (patho)physiologically relevant pHs (4.5–7.5), enabled assessment of both cuprous and cupric ions chelation and comparison of the relative affinities of the tested compounds for copper. In the case of potent chelators, the stoichiometry could be estimated too. Clioquinol, chloroxine and EDTA formed very stable complexes with Cu+/Cu2+ at all tested pHs, while copper complexes with trientine were stable only under neutral or slightly acidic conditions. Non-substituted 8-hydroxyquinoline was a less efficient copper chelator, but still unequivocally more potent than D-PEN. Both 8-hydroxyquinoline and D-PEN chelation potencies, similarly to that of trientine, were pH-dependent and decreased with pH. Moreover, only D-PEN was able to reduce cupric ions. Conclusively, BCS assay represents a rapid, simple and precise method for copper chelation measurement. In addition, lower binding affinity of D-PEN compared with 8-hydroxyquinolines and trientine was demonstrated.

Copper redistribution in murine macrophages in response to Salmonella infection

The movement of key transition metal ions is recognized to be of critical importance in the interaction between macrophages and intracellular pathogens. The present study investigated the role of copper in mouse macrophage responses to Salmonella enterica sv. Typhimurium. The copper chelator BCS (bathocuproinedisulfonic acid, disodium salt) increased intracellular survival of S. Typhimurium within primary mouse BMM (bone-marrow-derived macrophages) at 24 h post-infection, implying that copper contributed to effective host defence against this pathogen. Infection of BMM with S. Typhimurium or treatment with the TLR (Toll-like receptor) 4 ligand LPS (lipopolysaccharide) induced the expression of several genes encoding proteins involved in copper transport [Ctr (copper transporter) 1, Ctr2 and Atp7a (copper-transporting ATPase 1)], as well as the multi-copper oxidase Cp (caeruloplasmin). Both LPS and infection with S. Typhimurium triggered copper accumulation within punctate intracellular vesicles (copper 'hot spots') in BMM as indicated by the fluorescent reporter CS1 (copper sensor 1). These copper hot spots peaked in their accumulation at approximately 18 h post-stimulation and were dependent on copper uptake into cells. Localization studies indicated that the copper hot spots were in discrete vesicles distinct from Salmonella containing vacuoles and lysosomes. We propose that copper hot spot formation contributes to antimicrobial responses against professional intracellular bacterial pathogens.

硫酸銅電気めっき液中の一価銅の分析

Analysis of Cu(I) in copper sulfate electroplating solution was conducted by absorption of a chelate of Cu(I) with bathocuproinedisulfonic acid, disodium salt (BCS). Although the absorption of new copper sulfate electroplating solutions was negligible, the absorption of operating solutions was clear. The difference of new solutions and operating solutions was also verified using an electrochemical method for detection of Cu(I). We concluded that the Cu(I) ions are measurable using this chelate reagent. The absorption increased quickly in a few minutes after mixing with the chelate reagent and subsequently continued to increase slowly. To clarify this phenomenon, the organic compounds in the plating solutions were analyzed and Cu(I)-PEG (polyethylene glycol) complexes with different chain lengths were detected using MALDI-MS. Results show that Cu(I) ions exist in the plating solutions not only as small complexes with small organic compounds but also as large complexes with PEG. Small complexes of Cu(I) can react quickly with BCS and cause the rapid increase of the absorption in a few minutes after mixing. Cu(I)-PEG complexes prevent the chelating reaction of Cu(I) with BCS by steric hindrance of PEG, which explains the subsequent slow increase of the absorption. Using this chelate method, we monitored quantities of Cu(I) in copper sulfate electroplating production lines, detected the variation of Cu(I) quantities, and found the increase during the resting state of the lines.

Evaluation of the copper(II) reduction assay using bathocuproinedisulfonic acid disodium salt for the total antioxidant capacity assessment: The CUPRAC–BCS assay

There is heightened interest in determining antioxidant status of individuals in experimental and clinical studies investigating progression of diseases or diverse aspects of oxidative stress, among others. The aim of this study was to evaluate the copper(II) reduction assay with bathocuproinedisulfonic acid disodium salt as chelating agent (the CUPRAC–BCS assay) for the total antioxidant capacity (TAC) assessment in human plasma and urine. Samples from 20 individuals were determined with four spectrophotometric assays—CUPRAC–BCS, ferric reducing ability of plasma (FRAP), trolox equivalent antioxidant capacity (TEAC), and 1,1-diphenyl-2-picrylhydrazyl assay (DPPH)—to compare these methods. CUPRAC–BCS was significantly correlated with FRAP and TEAC for plasma and urine samples ( r > 0.5, P < 0.05 for all) and with DPPH for urine samples ( r = 0.925, P < 0.001) but not with DPPH for plasma samples ( r = 0.366, P = 0.112). However, the four methods do not agree given that lines of equality and regression were not matched up. The imprecision of the method is less than 6%, the detection limit is 41.8 μmol trolox equivalents/L, it is linear up to 2 mM trolox, and ethylenediaminetetraacetic acid dihydrate disodium salt (EDTA) binds to Cu(II), avoiding the formation of Cu(I)–BCS complex. This study shows that CUPRAC–BCS is a simple, fast, inexpensive, and suitable method for TAC assessment in human urine and heparinized plasma samples.

Formation of 8-hydroxydeoxyguanosine in calf thymus DNA treated with tert-butylhydroquinone, a major metabolite of butylated hydroxyanisole

Oxidative DNA damage caused by butylated hydroxyanisole (BHA), 2- tert-butyl(1,4)hydroquinone (TBHQ, a metabolite of BHA) and 2,5-di- tert-butyl(1,4)hydroquinone (DTBHQ), as well as 2,6-di- tert-butyl(1,4)benzoquinone (BHTQ, a metabolite of butylated hydroxytoluene), was evaluated by measuring the formation of 8-hydroxy-deoxyguanosine (80HdG) in calf thymus DNA. 80HdG formation was greatly increased by TBHQ in a concentration-dependent manner. This effect was strongly enhanced by CuCl 2 and suppressed by EDTA, bathocuproinedisulfonic acid disodium salt, methionine, glutathione reduced form or catalase, but was not affected by mannitol, sodium benzoate or sodium azide. Thus, TBHQ-induced 80HdG formation may be mediated by copper. DTBHQ also induced the formation of 80HdG, though to a much lesser extent than TBHQ, and its effect was stimulated by CuCl 2. BHA had a small enhancing effect at high concentration, only in the presence of CuCl 2, whereas in the case of BHTQ, it occurred both in the presence of CuCl 2 and FeCl 2.

Formation of 8-hydroxydeoxyguanosine in calf thymus DNA treated in vitro with phenylhydroquinone, the major metabolite of O-phenylphenol.

The generation of 8-hydroxydeoxyguanosine (8OHdG) in calf thymus DNA treated with O-phenylphenol (OPP) or its major metabolites, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), was studied. The content of 8OHdG residues was increased in DNA treated with PHQ, and the generation of 8OHdG was highly dependent on PHQ concentration. PBQ had little effect on the formation of 8OHdG, and OPP had no effect. The formation of 8OHdG by PHQ was reduced by oxygen radical scavengers such as catalase, sodium benzoate and sodium azide. The PHQ-induced 8OHdG formation was accelerated by the addition of CuCl or CuCl2 to the reaction mixture, but was decreased by the addition of chelating agents such as EDTA, bathocuproinedisulfonic acid disodium salt (bathocuproine disulfonate) and O-phenanthroline. These results demonstrate that hydroxyl radicals generated in the process of oxidation of PHQ contribute to the formation of 8OHdG in DNA, and copper ions facilitate the oxidative DNA damage. Copper ions greatly accelerated the PHQ-induced DNA cleavage in vitro, although they had no effect on cleavage without PHQ. On the other hand, DNA cleavage occurred by the addition of FeCl2 in the absence and presence of PHQ. FeCl2 stimulates 8OHdG formation only slightly with or without PHQ. Furthermore, the stimulatory effect of FeCl2 on 8OHdG formation was observed even in the presence of EDTA. The formation of 8OHdG in bladder DNA is likely to be one of a series of events leading to bladder tumors seen in rats fed OPP-containing diet.