An In Vitro Study of the Interaction of Copper(II) Salicylates with Genomic DNA Using Gel Electrophoresis and Gel Permeation Chromatography

This papers describes the in vitro interaction of copper(II) acetylsalicylate and copper(II) salicylate with genomic DNA isolated from human blood. The two drug substances were found to bind to DNA after incubation with whole blood over night. Bonding was confirmed by detection of separated DNA electrophoresis bands for copper, copper(II) acetylsalicylate, copper(II) salicylate, acetylsalicylic acid and salicylic acid. Drug–DNA interactions were observed during electrophoresis in the form of fragmentation by formation of two bands when compared to controls. Gel permeation chromatography parameters also confirmed the occurrence of fragmentation. The use of gel permeation chromatography parameters as a measure of fragmentation of DNA is discussed. The fragmentation of genomic DNA after incubation with copper(II) acetylsalicylate and copper(II) salicylate suggested that these drug substances might be responsible for cytotoxicity in vivo.     

The Comparison of Electrochemical Assay and Agarose Gel Electrophoresis for the Determination of DNA Damage Induced by Kainic Acid

A possible DNA damage after interaction of kainic acid (KA) with calf thymus double stranded DNA and genomic DNA was herein determined in in vitro and in vivo conditions using; electrochemical assay and agarose gel electrophoresis. The changes in guanine signal were detected as an indicator of DNA damage in genomic DNA samples isolated from 1 or 10 mg/kg KA‐treated animals. The decreased levels of guanine signal were found as 29% and 33% by 1 and 10 mg/kg KA treatment when compared to controls, respectively. The results of gel electrophoresis confirmed DNA damage obtained in identical samples by electrochemical method.     

Indirect Determination of some Analgesic-Inflammatory Drugs by AAS

ABSTRACT

An Indirect AAS method has been developed for the determination of analgesic-inflammatory drugs, mefenamic acid, flufenamic acid and diclofenac sodium. The method depends on the complexation with copper (II) ammine sulphate. The complex was extracted into chloroform and the concentrations of substances were determined indirectly by AAS measurement of copper, after re-extraction into 0.3 N nitric acid solution. The developed method was applied to the assay of the drug substances in commercial tablet formulations. The results were statistically compared with those obtained by HPLC method by t- and F-tests at 95% confidence level. Calculated t and F values were both lower than the table values.     

B → A Conformational Transition of DNA under the Influence of a Copper Thiolate,the Copper-cysteine: Biophysical,Spectroscopic and Gel Electrophoresis Studies

The interaction of calf-thymus DNA with copper(II)-cysteine (Cucys) was monitored by various biophysical methods. The interaction ratio was determined by the u.v.-spectrophotometric method, which was found to be 0.14. As the DNA 260 nm band remained insensitive followed by interaction with copper(II)-cysteine, the interaction ratio and the binding constant were determined by monitoring the 248 nm copper-cysteine charge-transfer (CT) band. Visible d–d spectra of Cucys proved that copper remained in the copper(II) state after interaction with DNA. The CD and thin film i.r. spectral studies revealed that there occurred B→A conformational transition of DNA when it was bound by copper-cysteine. Gel electrophoresis study confirmed the binding pattern.     

Separation and recovery of nucleic acids with improved biological activity by acid‐degradable polyacrylamide gel electrophoresis

One of the fundamental challenges in studying biomacromolecules (e.g. nucleic acids and proteins) and their complexes in a biological system is isolating them in their structurally and functionally intact forms. Electrophoresis offers convenient and efficient separation and analysis of biomacromolecules but recovery of separated biomacromolecules is a significant challenge. In this study, DNAs of various sizes were separated by electrophoresis in an acid‐degradable polyacrylamide gel. Almost 100% of the nucleic acids were recovered after the identified gel bands were hydrolyzed under a mildly acidic condition and purified using anion exchange resin. Further concentration by centrifugal filtration and a second purification using ion exchange column chromatography yielded 44–84% of DNA. The second conventional (non‐degradable) gel electrophoresis confirmed that the nucleic acids recovered from acid‐degradable gel bands preserved their electrophoretic properties through acidic gel hydrolysis, purification, and concentration processes. The plasmid DNA recovered from acid‐degradable gel transfected cells significantly more efficiently than the starting plasmid DNA (i.e. improved biological activity via acid‐degradable PAGE). Separation of other types of nucleic acids such as small interfering RNA using this convenient and efficient technique was also demonstrated.     

The preparation of nano‐scope chitosan‐oligomer copper complexes and their interaction with DNA

Water‐soluble low molecular weight chitosan of nanometer level and its copper complexes were prepared, and characterized by IR spectra, elemental analysis and gel permeation chromatography (GPC). The modes and mechanism of these copper complexes interaction with DNA were studied by a fluorescent probe method and electrophoresis analysis. It is suggested that there are electrostatic and intercalation modes of copper complexes interacting with DNA. At first, the cationic complex electrostaticly binds to the negatively charged phosphate backbone of DNA, and then a portion of the complex intercalates between the base pairs on the DNA duplex strand. Copyright © 2005 John Wiley & Sons, Ltd.     

Gel Permeation Chromatography of Polymers from Wood

Abstract

Polymers from wood, including cellulose via the methylol derivative, are soluble in dimethyl sulfoxide (DMSO). To perform gel permeation chromatography of these high polymers, a packing material was sought which combined adequate pore size with stability in DMSO. Contrary to recommended practice, it was discovered that gel permeation chromatography in DMSO can be performed on prepacked, high-performance columns of μ-Styragel without adverse effects from bead swelling. When using the methylol derivative of cellulose, it is necessary to freeze dry the reaction mixture to obtain a product which gives reproducible results from gel permeation chromatography.     

Extraction of lead(II) and copper(II) from salicylate media by tributylphosphine oxide.

Tributylphosphine oxide (TBPO) is proposed as an extractant for the extraction of lead(II) and copper(II) from salicylate media. The optimum conditions were evaluated by varying the experimental parameters, such as the pH, sodium salicylate concentration, tributylphosphine oxide (TBPO) concentration, shaking period and various diluents. The probable extracted species, deduced from log-log plots were Pb(HSal)2.2TBPO and Cu(HSal)2.2TBPO. The extraction took place through a solvation mechanism. The method permits the binary separation of lead(II) and copper(II) from commonly associated elements as well as the mutual separation of lead(II) and copper(II). The method is applicable to the determination of lead(II) and copper(II) in various alloys as well as environmental and pharmaceutical samples.     

Gel Permeation Chromatography of Insulin

Abstract

Aquas gel permeation chromatography of insulin under denaturating conditions has been successfully performed on three different chromatographic supports. The separation pattern was identical to that obtained on soft gels (Sephadex, BioGel). The elution time was 10-20 min, recovery 98-100%.     

Rapid quantification of DNA methylation by high performance capillary electrophoresis

The actual methods to evaluate total DNA methylation based on high performance liquid chromatography (HPLC) are long and tedious due to the specific running buffers required. In this work, a new open-tube capillary electrophoresis system has been applied to the separation of acid hydrolyzed genomic DNA and so, to the evaluation of genomic DNA methylation. Several running conditions were tested but separation of cytosine and 5-methyl-cytosine was only possible by sodium dodecyl sulfate (SDS) micelle system. The importance of sample dissolution preparation has also been demonstrated. The results of this study open up the possibility of quantification of the relative methylation degree of rapid genomic DNA by a simple method based on high performance capillary electrophoresis (HPCE).     

Gel permeation chromatography of polyamide by N-trifluoroacetylation

Successful chromatograms were obtained for trifluoroacetylated nylon 12 (N-TFA-N) in gel permeation chromatography (GPC) with dichloromethane as eluent. It was confirmed that the method of universal calibration was applicable for N-TFA-N and polystyrene. The average molecular weights of nylon 12 were obtained from the chromatogram of N-TFA-N by use of the calibration curve for polystyrene. The stability of N-TFA-N in solution was examined from various angles. Satisfactory results are obtained, if the sample solution is measured within 48 h after preparation.     

Extraction of cadmium,lead, cobalt,copper, and zinc ions from aqueous solutions into hydrophilic–hydrophobic ionic liquids

Extraction of cadmium(II), lead(II), cobalt(II), copper(II), and zinc(II) into ionic liquids tetraoctylammonium N-lauroyl sarcosinate and trioctylmethylammonium salicylate is studied. Cadmium(II), lead(II), copper(II) in tetraoctylammonium N-lauroyl sarcosinate and copper(II) in trioctylmethylammonium salicylate are quantitatively extracted from neutral and weakly alkaline solutions in the absence of additional reagents. The effect of the composition of aqueous and organic phases, as well as the contact time, on metal extraction is investigated.     

Copper(II)–Girard's T complex as a promising anti‐tumor agent

A copper(II) complex was evaluated for its anti‐tumor activity. Firstly, electrophoretic studies were applied on the complex. These studies revealed the binding of the complex to calf thymus DNA, leading to a delay in electrophoretic mobility of the DNA molecule. Secondly, spectroscopic data pointed out that the λ_max of DNA was shifted to a longer wavelength, which was accompanid by a hyperchromic shift. Moreover, the λ_max of copper(II) complex was shifted to a shorter wavelength. The favorable reaction conditions between the DNA molecule and the copper(II) complex were studied. Thirdly, The effects of the ligand and the Cu(II) ion were tested separately on the DNA molecule by electrophoresis technique. Furthermore, the fluorescence quenching of DNA bound ethidium ion by Cu(II)‐Girard's T complex was noticed. The IR spectral data of DNA before and after the reaction with the copper(II) complex indicated that the interaction takes place through the carbonyl group of DNA nucleobases. Finally, a significant increase in the mean survival of EAC (Ehrlich ascites carcinoma) tumor‐bearing mice was observed when treated with the copper(II) complex. The tumor volume was also significantly reduced (p < 0.0001). Electrophoretic studies showed that the DNA pattern extracted from EAC cells of tumor‐bearing mice was affected after treatment with the copper(II) complex. Flow cytometric studies showed that this complex may be taken into consideration in seeking novel anti‐tumor agents. Copyright © 2010 John Wiley & Sons, Ltd.     

Dose-dependent induction of apoptosis or necrosis in human cells by organotin compounds

In the present study, the mode of cell death induced by highly toxic trialkylated tin compounds has been evaluated. Treatment of undifferentiated HL-60 cells with submicromolar to micromolar concentrations of tri-n-butyltin (TBT) led only to a slight decrease in cell viability measured with trypan blue exclusion. Nevertheless, cell membrane blebbing was observed by means of light microscopy and condensation of nuclear chromatin and formation of apoptotic bodies was demonstrated in Hoechst 33342 stained cells. The nuclear chromatin condensation was associated with an extensive DNA fragmentation. Visualized by agarose gel electrophoresis, genomic DNA appeared as a characteristic ladder-like pattern of DNA fragments which is the biochemical hallmark of apoptosis. The typical internucleosomal DNA digestion was concentration-dependent and began within 2 to 3 h of incubation. During the incubation period a persistent and steady elevation of intracellular free calcium concentration ([Ca²⁺]_i) could be detected. Furthermore, the chromatin condensation and DNA fragmentation could be blocked by supplementation of the incubation medium with zinc pointing to an activation of a zinc-sensitive and calcium-dependent endogenous endonuclease. Higher concentrations of tributyltin (≥ 5 μmol/L TBT) led within hours to a cell killing with degenerative changes indicative of necrosis, demonstrated by plasma membrane disruption which was accompanied by random DNA breakdown. Furthermore, these concentrations also provoked a persistent elevation in [Ca²⁺]_i which reached, even after 10 min, higher levels in comparison with apoptosis-inducing concentrations. The loss in membrane integrity observed at these concentrations of TBT could be due to an activation of calcium-dependent phospholipases. Here it is shown that activation of cytosolic phospholipase A₂ (cPLA₂) leads to liberation of arachidonic acid (AA) out of the phospholipid membrane. The results presented here demonstrate that organometals are able to induce different cell death pathways depending on the applied concentration: low concentrations led to apoptosis whereas high concentrations stimulate necrosis. We suggest that there exists a direct correlation between the intracellular free calcium concentration and the mode of cell death. Received: 1 August 1997 / Revised: 8 October 1997 / Accepted: 10 October 1997     

Dose-dependent induction of apoptosis or necrosis in human cells by organotin compounds

In the present study, the mode of cell death induced by highly toxic trialkylated tin compounds has been evaluated. Treatment of undifferentiated HL-60 cells with submicromolar to micromolar concentrations of tri-n-butyltin (TBT) led only to a slight decrease in cell viability measured with trypan blue exclusion. Nevertheless, cell membrane blebbing was observed by means of light microscopy and condensation of nuclear chromatin and formation of apoptotic bodies was demonstrated in Hoechst 33342 stained cells. The nuclear chromatin condensation was associated with an extensive DNA fragmentation. Visualized by agarose gel electrophoresis, genomic DNA appeared as a characteristic ladder-like pattern of DNA fragments which is the biochemical hallmark of apoptosis. The typical internucleosomal DNA digestion was concentration-dependent and began within 2 to 3 h of incubation. During the incubation period a persistent and steady elevation of intracellular free calcium concentration ([Ca²⁺]_i) could be detected. Furthermore, the chromatin condensation and DNA fragmentation could be blocked by supplementation of the incubation medium with zinc pointing to an activation of a zinc-sensitive and calcium-dependent endogenous endonuclease. Higher concentrations of tributyltin (≥ 5 μmol/L TBT) led within hours to a cell killing with degenerative changes indicative of necrosis, demonstrated by plasma membrane disruption which was accompanied by random DNA breakdown. Furthermore, these concentrations also provoked a persistent elevation in [Ca²⁺]_i which reached, even after 10 min, higher levels in comparison with apoptosis-inducing concentrations. The loss in membrane integrity observed at these concentrations of TBT could be due to an activation of calcium-dependent phospholipases. Here it is shown that activation of cytosolic phospholipase A₂ (cPLA₂) leads to liberation of arachidonic acid (AA) out of the phospholipid membrane. The results presented here demonstrate that organometals are able to induce different cell death pathways depending on the applied concentration: low concentrations led to apoptosis whereas high concentrations stimulate necrosis. We suggest that there exists a direct correlation between the intracellular free calcium concentration and the mode of cell death. Received: 1 August 1997 / Revised: 8 October 1997 / Accepted: 10 October 1997     

Studies on the metabolism of the α-pyrrolidinophenone designer drug methylenedioxy-pyrovalerone (MDPV) in rat and human urine and human liver microsomes using GC-MS and LC-high-resolution MS and its detectability in urine by GC-MS

Since the late 1990s, many derivatives of the α-pyrrolidinophenone (PPP) drug class appeared on the drugs of abuse market. The latest compound was described in 2009 to be a classic PPP carrying a methylenedioxy moiety remembering the classic entactogens (ecstasy). Besides Germany, 3,4-methylene-dioxypyrovalerone (MDPV) has appeared in many countries in Europe and Asia, indicating its worldwide importance for forensic and clinical toxicology. The aim of the presented work was to identify the phase I and II metabolites of MDPV and the human cytochrome-P450 (CYP) isoenzymes responsible for its main metabolic step(s). Finally, the detectability of MDPV in urine by the authors' systematic toxicological analysis (STA) should be studied. The urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified after work-up by GC-MS and liquid chromatography (LC)-high-resolution MS (LC-HR-MS). The studies revealed the following phase I main metabolic steps in rat and human: demethylenation followed by methylation, aromatic and side chain hydroxylation and oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Using LC-HR-MS, most metabolite structures postulated according to GC-MS fragmentation could be confirmed and the phase II metabolites were identified. Finally, the formation of the initial metabolite demethylenyl-MDPV could be confirmed using incubation of human liver microsomes. Using recombinant human CYPs, CYP 2C19, CYP 2D6 and CYP 1A2 were found to catalyze this initial step. Finally, the STA allowed the detection of MDPV metabolites in the human urine samples.     

Gel Permeation for the On Line Removal of Proteins from Plasma Samples Prior to HPLC

Abstract

Gel permeation chromatography (GPC) has been investigated for the on line removal of proteins from plasma samples prior to their analysis by HPLC. The results show that GPC is a mild and effective way to remove proteins from plasma samples. It can very well be coupled on line to HPLC, providing the solutes are suitable for preconcentration on the analytical column itself or on a small pre-column, after the GPC. Under these conditions excellent reproducibility and accuracy can be obtained.     

The coulometric determination of salicylate in serum

Constant-current coulometry can be used for the determination of salicylate in 100 μg of blood serum. The titration is preceded by extraction of acidified serum with ethylene dichloride followed by removal of the salicylate to an aqueous solution of pH 7.5 tris(hydroxymethyl)aminomethane buffer which also contains copper(II) sulfate to enhance recovery. A residual titration with bromine as titrant followed by addition of potassium thiocyanate is used because of the slow bromination of salicylate. Serum samples containing salicylate in the range 4–60 mg/100 ml were analyzed. Recoveries of about 90% were obtained.     

Calibration in High Temperature Gel Permeation Chromatography of Linear Polyethylenes

Abstract

The mathematical relationship between the gel permeation chromatography calibration curves of polystyrene and linear polyethylene has been determined in 1,2,4-trichlorobenzene at 130, 135 and 140°C. The experimentally determined relationship is in good agreement with that predicted from application of the principles of the universal calibration technique and published Mark-Houwink coefficients. Definition of this relationship enables the use of polystyrene as a secondary standard for gel permeation chromatographic determination of linear polyethylene molecular weight determinations.