Dithiothreitol-Lucigenin Chemiluminescent System for Ultrasensitive Dithiothreitol and Superoxide Dismutase Detection.

1,4-Dithiothreitol (DTT), a highly water-soluble and well-known reducing agent for preservation and regeneration of sulfhydryl groups in biomedical applications, has been developed as an efficient and stable coreactant of lucigenin for the first time. DTT efficiently reacts with lucigenin to generate intense chemiluminescence (CL), eliminating the need for external catalysts to facilitate the lucigenin CL. The DTT-lucigenin CL is approximately 15-fold more intense when compared with the lucigenin-H2O2 classical system. Superoxide dismutase (SOD) remarkably quenches the DTT-lucigenin CL. Based on this phenomenon, a newly developed CL approach for the determination of SOD was proposed with a linear range of 0.01-1.5 µg/mL and a limit of detection of 2.2 ng/mL. Various factors affecting the CL emission of the DTT-lucigenin probe were studied and optimized. Plausible mechanistic pathways for the CL coreaction of lucigenin with DTT were proposed and fully discussed. Our proposed method not only has the merit of being selective toward the target analytes but also eliminates the need for the complex synthesis of luminescent probes and facilitates the sensitive detection of SOD in human serum and cosmetics SOD raw material with satisfactory recoveries.

3-[(E)-(acridin-9'-ylmethylidene)amino]-1-substituted thioureas and their biological activity.

This paper describes the synthesis of a novel series of acridine thiosemicarbazones through a two-step reaction between various isothiocyanates and hydrazine followed by treatment with acridin-9-carbaldehyde. The properties of this series of seven new derivatives are studied using NMR and biochemical techniques, and the DNA-binding properties of the compounds are determined using spectrophotometric studies (UV-vis absorption, fluorescence, and circular/linear dichroism) and viscometry. The binding constants K are estimated as being in the range of 2.2 to 7.8×104M-1 and the percentage of hypochromism was found to be 22.11-49.75% (from UV-vis spectral titration). Electrophoretic experiments prove that the novel compounds demonstrate moderate inhibitory effects against Topo I activity at a concentration of 60×10-6M.

Evaluation of a general toxicity study incorporating phototoxicity assessments in Sprague-Dawley rats.

Previously, we showed that phototoxicity assessments in Sprague-Dawley (SD) rats can detect phototoxic potential to the same degree as those in guinea pigs. In this study, we examined whether phototoxicity assessments can be incorporated into general toxicology studies, using SD rats. Three phototoxic compounds were tested. Acridine and 8-methoxypsoralen (8-MOP) were transdermally administered, and 8-MOP and lomefloxacin were orally administered. The animals were allocated to three groups for each compound: single-dose, repeated-dose, and repeated-dose plus toxicokinetics (TK). The single-dose group was irradiated with UV-A and UV-B after a single administration of the drug. The repeated-dose and TK groups were irradiated after 8 days of repeated administration of the drug. Blood samples were also collected from the TK group on days 1 and 7 after administration. The phototoxic compounds resulted in skin reactions in all the groups, with no difference in the degree of skin reaction among the three groups. In the TK measurements, all of the phototoxic compounds were detected in the plasma samples, and the irradiation timing was close to the Tmax. These results indicate that phototoxic potential could be evaluated in the TK group, and phototoxicity assessments could be incorporated into general toxicology studies. This reduces the number of studies and animals required, thus shortening the research and development period, and supporting the 3Rs principle of animal experiments. The study also provides information regarding appropriate irradiation timings, differences between the sexes, and dose-response, in turn enabling the phototoxic risk of the compounds to be clearly evaluated.

The Crystal Structure and Behavior of Fenamic Acid-Acridine Complex Under High Pressure.

The crystal structure of fenamic acid-acridine complex is determined by X-ray diffraction. The strong OHN hydrogen bond linking the complex components and other interactions responsible for packing of the molecules into a crystal are investigated within the Quantum Theory of Atom in Molecule theory. The crystal structure is compared with the structure optimized at B3LYP/6-311++G** level and with the theoretical structures optimized under systematically changed pressure. Analysis of the lattice constants, hydrogen bond lengths, and angles of the inter- and intramolecular hydrogen bond under compression is performed. The structural transformation observed at 5 GPa is connected with a change in the intermolecular OHN hydrogen bond. The proton shifts to acceptor and a new interaction in the crystal appears.

Whole-Body Distribution and Radiation Dosimetry of 11C-Elacridar and 11C-Tariquidar in Humans.

UNLABELLED: (11)C-elacridar and (11)C-tariquidar are new PET tracers to assess the transport activity of P-glycoprotein (adenosine triphosphate-binding cassette subfamily B, member 1 [ABCB1]) and breast cancer resistance protein (adenosine triphosphate-binding cassette subfamily G, member 2 [ABCG2]). This study investigated the whole-body distribution and radiation dosimetry of both radiotracers in humans. METHODS: Twelve healthy volunteers (6 women, 6 men) underwent whole-body PET/CT imaging over the 90 min after injection of either (11)C-elacridar or (11)C-tariquidar. Radiation doses were calculated with OLINDA/EXM software using adult reference phantoms. RESULTS: Biodistribution was consistent with a major elimination route of hepatobiliary excretion, which may be mediated by ABCB1 and ABCG2. High radioactivity uptake was seen in liver, followed by spleen and kidneys, whereas brain uptake was lowest. Effective doses were 3.41 ± 0.06 µSv/MBq for (11)C-elacidar and 3.62 ± 0.11 µSv/MBq for (11)C-tariquidar. CONCLUSION: Our data indicate that both (11)C-elacridar and (11)C-tariquidar are safe radiotracers, for which an injected activity of 400 MBq corresponds to a total effective dose of approximately 1.5 mSv.

C60-SIMS imaging of nanoparticles within mammalian cells.

To achieve successful drug delivery via nanoparticles the interactions between the nanoparticle and the chemistry of the surrounding biological environment is of central importance. A thorough understanding of these interactions is necessary in order to better elucidate information regarding drug pathways and mechanisms of action in treatment protocols. As such, it is important to identify the location of the nanoparticle, the state of its functionalization, as well as any changes in the cellular environment. The use of cluster secondary ion mass spectrometry (SIMS) using C60 (+) primary ions makes simultaneous acquisition of this information possible. Here, SIMS has been successfully used to chemically image gold nanoparticles (AuNPs) within a model, single cell system involving macrophage-like RAW 264.7 cells. The macrophage-like properties of this cell line make it extremely well-suited for cell-uptake studies. Both AuNPs and two pharmaceutical compounds, amiodarone and elacridar, were successfully imaged within a cellular system using cluster SIMS. To verify that SIMS can also be used to detect functionalization and nanoparticles simultaneously, fluorophore-functionalized AuNPs were studied as a model system. The fluorescent characteristics of these functionalized nanoparticles enabled the visual confirmation of the presence and location of the particles within the cell.

Development of a highly sensitive chemiluminescent assay for hydrogen peroxide under neutral conditions using acridinium ester and its application to an enzyme immunoassay.

We developed a highly sensitive chemiluminescent (CL) assay for hydrogen peroxide using 10-methyl-9-(phenoxycarbonyl) acridinium fluorosulfonate (PMAC) that produced chemiluminescence under neutral conditions and applied it to an enzyme immunoassay (EIA). One picomole of hydrogen peroxide could be detected using the optimized PMAC-CL method and 6.2 × 10(-20) mol ß-D-galactosidase (ß-gal) could be detected by combining an indoxyl derivative substrate and the proposed PMAC-CL method. This highly sensitive CL ß-gal assay was applied to an EIA for thyroid-stimulating hormone (TSH) using ß-gal as a label enzyme; 0.02-100.0 µU/mL TSH in human serum could be assayed directly and with high reproducibility.

Investigating the binding of acridine, acridine orange, and acridine yellow G to humic acid through fluorescence quenching.

A fluorescence quenching method was used to determine the equilibrium binding constants for the association of acridine, acridine orange, and acridine yellow G to humic acid. The fluorescence of each polycyclic aromatic nitrogen heterocycle (PANH) was monitored as aliquots of humic acid were added, and a Stern-Volmer plot was produced in which the slope is the equilibrium constant of the binding reaction. The quenching experiments were performed at temperatures of 30, 35, 40, and 45 °C. A van't Hoff plot generated from the equilibrium binding constants as a function of temperature for a given PANH resulted in a linear plot. Calculation of the ΔHbinding, ΔGbinding, and ΔSbinding for each PANH leads to the conclusion that the equilibrium binding constant, and ΔGbinding, may be predictors of bioavailability. The other thermodynamic quantities, ΔHbinding and ΔSbinding, are helpful in understanding the relative binding of the compounds. For example, acridine yellow G appears to be the least bioavailable of the three PANHs studied because of its strong ΔHbinding = -29.8 kJ/mol, which leads to ΔGbinding = -0.71 kJ/mol. While acridine orange and acridine have similar ΔHbinding values, acridine orange is more likely to bind to humic acid because the ΔSbinding for the process is less negative. Thermodynamic values and equilibrium binding constants for all three compounds are reported.

A new liquid chromatography-tandem mass spectrometry method using atmospheric pressure photo ionization for the simultaneous determination of azaarenes and azaarones in Dutch river sediments.

A new method for the analysis of azaarenes and their degradation products (azaarones) was developed, optimized and validated using liquid chromatography coupled with atmospheric pressure photo ionization tandem mass spectrometric detection (LC-APPI/MS/MS). Seventeen compounds including 4 PAHs (naphthalene, anthracene, phenanthrene, benz[a]anthracene), 7 azaarenes (quinoline, acridine, phenanthridine, 5,6-benzoquinoline and 7,8-benzoquinoline, benzo[a]acridine, benzo[c]acridine), and 6 azaarones (2-OH-quinoline, 4-OH-quinoline, 5-OH-quinoline, 6-OH-quinoline, 9(10H)-acridone, 6(5H)phenanthridinone) were analyzed in sediment samples from Dutch rivers. All compounds were analyzed simultaneously in multi reaction monitoring (MRM) mode. Soxhlet extraction was used for the extraction of analytes from sediments. The limits of quantification of azaarenes and azaarones varied from 0.21 to 1.12µg/l and from 0.23 to 1.58µg/l, respectively. The limits of quantification for PAHs varied from 32 to 769µg/l. Matrix-independent recoveries of sediment samples were in the range 85-110%; matrix-dependent recoveries were in the range 73-148%, respectively. The method was tested on real sediment samples and the results were compared with a previous study in which GC/MS/MS was used for the simultaneous measurement of azaarenes and azaarones. 4-, 5- and 6-OH-quinolines and naphthalene, anthracene and phenanthrene were not present or below detection limits in some samples. All other analytes were present in samples in the concentration range 0.2-1200ng/g (dw). To our knowledge, this is the first report showing the possibility of measurement non-polar polyaromatic hydrocarbons together with polar azaarenes and their degradation products azaarones simultaneously with sufficient sensitivity and accuracy using LC/MS/MS.

The effect of magnesium and vitamin E pre-treatments on irradiation-induced oxidative injury of cardiac and pulmonary tissues in rats: a randomized experimental study.

OBJECTIVE: The aim of this study was to investigate the effect of pre-treatment with the free radical scavenging molecules, magnesium and vitamin E, on lipid peroxidation to limit radiation-induced heart and lung injury. METHODS: Female Sprague-Dawley rats were divided into 4 groups by a simple randomization method as saline-treated control (n=4), saline-treated irradiated (IR; n=6), magnesium sulphate-treated irradiation (IR) (Mg+IR; n=6) and vitamin E-treated IR (vit E+IR; n=6), respectively. The animals were given either saline, Mg (600 mg/kg/day) or vit E (100 mg/kg/day) intraperitoneally for five days prior to irradiation. Twelve hours after the fifth injection, animals in irradiation groups were irradiated to 20 Gy using 6 MV photons in linear accelerator. Twenty-four hours later cardiac and lung tissue samples were obtained for determination of myeloperoxidase activity (MPO), malondialdehyde (MDA) levels, and luminol and lucigenin levels measured by chemiluminescence (CL) methods. RESULTS: No significant changes were observed between cardiac and pulmonary MDA and CL results of the experimental groups. However, cardiac and pulmonary MPO activities in the saline-treated IR group were increased as compared to control group (p<0.05 for all), while in the Mg-pretreated and vit E pretreated groups neutrophil infiltration was reduced, reaching to statistical significance only in the Mg-pretreated group (p<0.05). CONCLUSION: Prophylactic use of magnesium sulfate has limited the infiltration of neutrophils to both the cardiac and pulmonary tissues at the early 24 h of irradiation. However, how limiting neutrophils as the sources of free radicals and inflammatory mediators would alter oxidative stress of heart and lung tissues in the long-term is not clear yet.

Chemiluminescent reductive acridinium triggering (CRAT)--mechanism and applications.

Acridinium esters traditionally are triggered using basic hydrogen peroxide. By serendipity, we have found that acridinium esters can also be triggered with emission of chemiluminescence by reductive triggering, e.g., by zinc metal or reduced forms of ferric and cupric salts. Furthermore, organic reducing compounds like dithiothreitol, tricarboxyethylphosphine or glutathione could be used in combination with organic oxidants like quinones or inorganic ferric or cupric salts. Mechanisms are proposed which involve the intermediacy of superoxide. Two forms of reactive oxygen species (i.e., hydrogen peroxide and superoxide) could be discriminated based on differences in kinetics. Some applications (improved detection of acridinium ester, use of acridinium ester as redox probes) are discussed.

Presence and ex vivo formation of acridone in blood of patients routinely treated with carbamazepine: exploration of the 9-acridinecarboxaldehyde pathway.

Carbamazepine (CBZ) is a useful anticonvulsive drug associated with rare severe adverse drug reactions. The physio-pathological mechanisms of these reactions are unknown although evidence of immunological activation has been reported. The ability of 9-acridinecarboxaldehyde, a CBZ metabolite, to interact with leukocyte constituents was demonstrated, and catabolism of this compound into acridine (AI) and acridone (AO) was observed in vitro. In this study, we have assessed ex vivo the role of the extra-hepatic 9-acridinecarboxaldehyde pathway in the metabolism of CBZ. First, we verified the presence of the terminal metabolites AI and AO in CBZ-treated patients. Then, we tested ex vivo the transformation of CBZ, epoxy CBZ, iminostilbene, and AI into AO in the blood of these patients. We observed no direct formation of hydroxylated CBZ metabolites in isolated blood, and CBZ did not react with blood cells. Conversely, we detected a dose-dependent transformation of epoxy CBZ, iminostilbene, and AI into AO with individual variations from patient to patient. AO might thus be considered as a metabolite of 9-acridinecarboxaldehyde that does not react with cells (detoxicant pathway) as well as a marker of the formation of toxic AI derivatives (toxicant pathway).

Challenge in the speciation of nitrogen-containing compounds in heavy petroleum fractions by high temperature comprehensive two-dimensional gas chromatography.

Extending the knowledge related to nitrogen-containing compounds presents an important interest for the petroleum industry due to their implication in atmosphere pollution as well as their inhibitive or refractive behaviour towards hydroprocessing. Most of the nitrogenated species are concentrated in heavy petroleum cuts. As no analytical method is resolutive enough for these heavy cuts, particularly regarding nitrogen-containing compounds, a new approach is needed. For this reason, this study focuses on the development of a GC×GC technique, through the hyphenation of a specific NCD detector with a GC×GC system at high temperature. The performances of highly polar thermally stable stationary phases, in particular those composed of promising ionic liquids, were monitored in normal and reversed configurations. Subsequently, after the extraction of neutral or basic compounds by adsorption on an ion-exchange resin, a first quantitative determination was attempted for a straight-run and a direct coal liquefaction vacuum gas oils. This study led to a better understanding of the behaviour of highly aromatic N-compounds by 2D-GC including an ionic liquid phase as well as a deeper N-characterization of heavy petroleum fractions.

Live-cell one- and two-photon uncaging of a far-red emitting acridinone fluorophore.

Total synthesis and photophysical properties of PENB-DDAO, a photoactivatable 1,3-dichloro-9,9-dimethyl-9H-acridin-2(7)-one (DDAO) derivative of a far-red emitting fluorophore, are described. The photoremovable group of the DDAO phenolic function comprises a donor/acceptor biphenyl platform which allows an efficient (> or = 95%) and rapid (< 15 micros time-range) release of the fluorescent signal and displays remarkable two-photon uncaging cross sections (delta(a) x Phi(u) = 3.7 GM at 740 nm). PENB-DDAO is cell permeable as demonstrated by the triggering of cytoplasmic red fluorescent signal in HeLa cells after one-photon irradiation (lambda(exc) around 360 nm) or by the generation of a red fluorescent signal in a delineated area of a single cell after two-photon photoactivation (lambda(exc) = 770 nm).

Soft-modeling based spectrofluorimetric study of simultaneous equilibria.

A two-way soft resolution method will fail when applied to a simultaneous equilibria system due to rank deficiency in its concentration profiles. Increasing the dimensionality of measurements from two-way to three-way data can be used to overcome this problem. Simultaneous dissociation of two weak acids is considered as a model for simultaneous equilibria. Three-way data obtained from excitation-emission spectrofluorimetric monitoring of a pH-metric titration is analyzed using a proper combination of well-known soft-modeling methods. Multivariate curve resolution-alternating least squares is used for calculating the excitation and emission spectral profiles of involved species and rank annihilation factor analysis for obtaining the contribution of each species in measured excitation-emission matrices at different pHs. The results of simulated and real simultaneous acids dissociation equilibria showed that the proposed combined method performs well even in situation when the equilibrium constants are close to each other. The applicability of method for study of an acidic dissociation is also shown.

beta-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG.

beta-Galactosidase (beta-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) beta-d-galactopyranoside (DDAOG), can be cleaved by beta-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a beta-gal activity assay method. The DDAO signal was stable for at least 18h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the beta-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The beta-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The beta-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of beta-gal systems currently in use.

Cell adhesion markedly increases lucigenin-enhanced chemiluminescence of the phagocyte NADPH oxidase.

Lucigenin-enhanced chemiluminescence (LECL) is widely used for the detection of reactive oxygen species released from various cells and mitochondria. However, the LECL response varies depending on cell species and assay conditions at least in part by unknown factors. Here we report that cell adhesion is an important factor for increasing LECL of tetradecanoylphorbol acetate (TPA)-stimulated human neutrophils. More than 90% LECL remained even after complete removal of the cell suspension 10 min after TPA stimulation, and approximately 22.5% of neutrophils were adhered to the reaction tube. These results indicate that LECL by an adhering neutrophil is approximately 45x higher than that by a non-adhering neutrophil. LECL by leukocyte adhesion deficiency neutrophils was one-fifth of that by normal neutrophils and completely disappeared when the cell suspension was removed, confirming that LECL depends highly on cell adhesion. The oxidase activity of adhering neutrophils measured after permeabilization with Renex 30 together with NADPH addition was similar to that of non-adhering neutrophils, indicating that lucigenin and cell adhesion do not enhance the oxidase activity. Based on these findings, we propose that a mixture of adhering and non-adhering neutrophils can be used for simultaneous screenings of adhering activity and the oxidase activity of neutrophils.

Vitamin C depletion increases superoxide generation in brains of SMP30/GNL knockout mice.

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(-)]. At 4 and 8 weeks, VC levels in brains from VC(-) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(-) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.

Chronic exposure of the oligochaete Lumbriculus variegatus to polycyclic aromatic compounds (PACs): bioavailability and effects on reproduction.

This study aimed to monitor PAC availability to the oligochaete Lumbriculus variegatus during 28 days of exposure to spiked sediments, in order to obtain reliable chronic effect concentrations for reproduction. Sediment toxicity tests were performed using three pairs of PAC isomers: two homocyclic compounds (anthracene and phenanthrene), two azaarenes (acridine and phenanthridine), and the two main transformation products of the azaarenes (acridone and phenanthridone). During the experiment, available PAC concentrations in pore water (estimated using solid phase microextraction) decreased more than total PAC concentrations in the sediment. Relating effect concentrations to PAC concentrations in pore water and in organisms showed that the two homocyclic compounds caused narcotic effects during chronic exposure, but only one of the four tested heterocyclic PACs caused narcotic effects. The transformation product phenanthridone was not toxic at the tested concentrations (up to 4000 micromol/kg dry sediment), whereas EC50 values for the parent compound phenanthridine and the isomer acridone were below the estimated limit for narcosis, suggesting a specific mode of action. These results demonstrated the unpredictable (isomer) specific toxicity of azaarenes and their transformation products, emphasizing the need of chronic toxicity testing to gain insight into the long-term effects of heterocyclic PACs, which have been overlooked in risk assessment.

Differentiation of heterocyclic regioisomers: a combined tandem mass spectrometry and computational study of N-acridin-4-ylbenzylamide and N-acridin-2-yl-benzylamide.

Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) has been used to differentiate two positional isomers of acridine derivatives, N-acridin-4-ylbenzylamide and N-acridin-2-ylbenzylamide. The study revealed that the isomeric ion structures produced by these heterocycles could be distinguished upon collision-induced dissociations (CID). In particular, the loss of a water molecule was shown to be a regiospecific reaction of the protonated N-acridin-4-ylbenzylamide, in which the location of the benzylamide substituent with respect to the acridinic nitrogen greatly assists proton migration by allowing the creation of intramolecular hydrogen bonds. To a lesser extent, the two isomers could also be distinguished by the difference in the abundance of the benzoyl cation in the MS/MS spectra of the [M+H]+ ions, as this ion is produced with a much higher rate from N-acridin-4-ylbenzylamide. Calculations based on quantum-mechanical models have been performed to evaluate the stability of the ion structures and to support mechanisms proposed for these two dissociation reactions.