LC-APCI(-)-MS Determination of 1-Chloro-2,4-dinitrobenzene, a Model Substrate for Glutathione S-Transferases.

1-Chloro-2,4-dinitrobenzene (CDNB) is widely used as a model substrate for measuring the enzyme activity of glutathione S-transferases in toxicity studies and in studies focusing on the metabolic capacity of different test systems. To allow the quantification of CDNB at low, nontoxic concentrations, we developed a sensitive liquid chromatography-mass spectrometry (LC-MS) technique, which is based on electron capture ionization using atmospheric pressure chemical ionization (APCI) in negative ion mode. Gas-phase reactions occurring under atmospheric pressure produce specific ions that allow direct CDNB quantification down to 17 ng/mL in water. Using the new technique, we were able to verify CDNB exposure concentrations applied in two typical toxicity studies with early life stages of the common model organisms, zebrafish (Danio rerio) and a zebrafish embryonic cell line (PAC2).

Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo.

Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation. The result, MitoCDNB, is taken up by mitochondria where it selectively depletes the mitochondrial GSH pool, catalyzed by glutathione S-transferases, and directly inhibits mitochondrial TrxR2 and peroxiredoxin 3, a peroxidase. Importantly, MitoCDNB inactivates mitochondrial thiol redox homeostasis in isolated cells and in vivo, without affecting that of the cytosol. Consequently, MitoCDNB enables assessment of the biomedical importance of mitochondrial thiol homeostasis in reactive oxygen species production, organelle dynamics, redox signaling, and cell death in cells and in vivo.

Synthesis of a new magnetic-MIP for the selective detection of 1-chloro-2,4-dinitrobenzene, a highly allergenic compound.

Molecularly imprinted polymers (MIPs) in combination with magnetic nanoparticles, in a core@shell format, were studied for selective detection of 1-chloro-2,4-dinitrobenzene (CDNB), a powerful allergenic substance. Magnetic nanoparticles were prepared by the co-precipitation method and mixed with oleic acid (OA). This material was then encapsulated in three types of hydrophobic polymeric matrix, poly-(MA-co-EDGMA), poly-(AA-co-EDGMA), and poly-(1-VN-co-EDGMA), by the mini-emulsion method. These matrices were used due to their ability to interact specifically with the functional groups of the analyte. Finally, the MIP-CDNB was obtained on the magnetic-hydrophobic surfaces using precipitation polymerization in the presence of the analyte. XRD diffraction patterns suggested the presence of magnetite in the composite and SEM analysis revealed a nanoparticle size between 10 and 18nm. Under the optimized adsorption conditions, the magnetic-MIP material showed a higher adsorption capacity (5.1mgg-1) than its non-magnetic counterpart (4.2mgg-1). In tests of the selectivity of the magnetic-MIP towards CDNB, α-values of 2.5 and 10.4, respectively, were obtained for dichlorophenol and o-nitrophenol, two structurally similar compounds, and no adsorption was observed for any other non-analogous analyte. The magnetic-MIP and magnetic-NIP were applied using water enriched with 0.5mgL-1 of CDNB, achieving recovery values of 83.8(±0.8)% and 66(±1)%, respectively, revealing the suitability of the material for detection of CDNB.

Simultaneous determination of 1-chloro-2,4-dinitrobenzene, 2,4-dinitrophenyl-S-glutathione and its metabolites for human placental disposition studies by high-performance liquid chromatography.

We developed and validated an HPLC method for determination of 1-chloro-2,4-dinitrobenzene (CDNB) and its glutathione conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG) to study the kinetics and mechanisms involved in DNP-SG formation and efflux, as a probe for human placental metabolism and transport. This method combines use of 3 microm solid phase, rapid mobile phase gradient with dual wavelength ultraviolet detection to permit determination of a lipophilic parent compound and its hydrophilic metabolites in a single short run. The selectivity, linearity, accuracy, precision, relative recovery and stability of the assay are sufficient for determining CDNB, DNP-SG and its metabolites from buffer and tissue samples to support placental drug metabolism and transport studies.

Peptide-binding assessment using mass spectrometry as a new screening method for skin sensitization.

Skin sensitization potential of low molecular weight chemicals was assessed by analyzing peptide-conjugate formation. Chemicals were incubated with a peptide, glutathione, and resultant mixtures were analyzed by mass spectrometry. Eighteen chemicals were assessed, and new peaks corresponding to chemical-peptide conjugates were detected for 13 of 14 known sensitizers. Conjugates were not detected for 4 negative chemicals. The method has advantages as a simple screening assay for assessing the sensitization potential of chemicals.

[Determination of 2,4-dinitrochlorobenzene in environmental water by primary-secondary wavelengths new spectrophotometry].

In basic solution, dinitrochlorobenzene may react on pyridine to form a purplish red compound. In this paper with the absorption property of a suspended liquid against light a new analytical method as primary-secondary wavelengths spectrophotometry was studied to determine trace dinitrochlorobenzene in environmental water. In 0-50 microg dinitrochlorobenzene/25 mL, the calculation curve is very stable and hardly affected by environment conditions. Experiments for practical waters have shown that the detection limit was 0.7 microg/25 mL dinitrochlorobenzene and RSD < or = 2.7% and the recovery rate was 99.2%-101.0%.

Reduction of MTT by glutathione S-transferase.

The reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to a blue formazan product is widely used for assaying cell survival and proliferation. The reduction reaction is catalyzed by dehydrogenases localized in the mitochondria of viable cells. As part of an analysis of the ability of glutathione S-transferase (GST) enzymes to protect cells from electrophilic compounds, we found extremely high background levels of the formazan product produced by cells that overexpressed the mouse GST P1-1 enzyme. Further analysis with purified GST enzymes confirmed the ability of these enzymes to reduce MTT in vitro. These data suggest that cytotoxicity assays using MTT should be interpreted with caution, especially when studying the effects of compounds that can influence GST expression.

Avaliaçäo da hipersensibilidade ao dinitroclorobenzeno em ratos tratados ou näo com ciclosporina / Evaluation of hipersensibility to dinitrochlorobenzeno in rats treated or not with cyclosporin-A

Avaliou-se nesse trabalho a resposta imunológica de ratos Wistar, através da hipersensibilidade tipo IV, celular ou retardada, provocada pelo DNCB em cinco grupos de 10 ratos. Para a imunizaçäo, aplicou-se sobre a pele dos animais 50 µl de DNCB, e a reaçäo foi desencadeada com concentraçöes crescentes de 10, 20, 30, 50 e 100 mg/ml na superfície da orelha esquerda e, após 24 horas, foi feita uma relaçäo de peso das orelhas esquerda/direita (controle). Todos os animais desenvolveram reaçöes de hipersensibilidade tipo IV num mesmo padräo clínico. O mesmo teste foi aplicado em cinco grupos de 5 ratos que receberam doses de 5, 7, 10, 30 e 50 mg/kg/dia de ciclosporina. Todos os animais tratados näo responderam ao teste, deduzindo-se que eles estavam imunossuprimidos pela ciclosporina

Dietary exposure of man to chlorinated benzenes in the United Kingdom.

Chlorinated benzenes are produced in large quantities on a world-wide basis. They are important industrial chemicals, chemical intermediates and components of the waste products of chemical synthesis. Their widespread occurrence and persistence, particularly in the case of the more highly chlorinated benzenes, has resulted in residues in food and human tissues. Information currently available about the levels of residues of these compounds in food has been summarised with particular reference to U.K. data. Estimation of average dietary intake is only possible for the most highly chlorinated compound, hexachlorobenzene, and is calculated to be 0.2 micrograms per person per day. Though insufficient data exist to assess dietary intake of other chlorinated benzenes in the U.K. it is considered unlikely that they would occur at levels greater than hexachlorobenzene in the diet.

Contaminants of dinitrochlorobenzene.

DNCB (2,4-dinitrochlorobenzene) is used in the treatment of alopecia areata, recalcitrant verrucae, and for evaluating immunocompromised patients. Currently, there is no pharmaceutical grade of DNCB available for clinical use. Recently, no nitrochlorobenzene contamination was found with the use of positive ion detection gas chromatography--mass spectrometry. We examined six commercial sources of DNCB, with the use of negative ion detection gas chromatography--mass spectrometry, for volatile impurities such as nitrochlorobenzene which might remain from the manufacturing process. The use of negative ion detection increases the sensitivity of this technic to benzene rings substituted with electron withdrawing groups like the nitrochlorobenzenes. We found that all sources of DNCB contain various combinations of nitrochlorobenzene, dinitrobenzene, nitrodichlorobenzene, and other isomers of DNCB. Nitrochlorobenzene has been shown to be mutagenic in the Ames test and carcinogenic in animal studies. The presence of nitrochlorobenzene and other contaminants in commercial grades of DNCB raises questions about the continued clinical application of this agent.

The role of mononitrochlorobenzene as a contaminant in dinitrochlorobenzene.

Dinitrochlorobenzene (DNCB) has been purported to possess potential mutagenic and carcinogenic hazards. This potential hazard may be due to contamination of DNCB with known carcinogenic precursors, mononitrochlorobenzenes. Three samples of commercially available DNCB were analyzed for the presence of such mononitrochlorobenzenes, which were absent in all samples tested. The implications of product contamination with mutagenic and/or carcinogenic impurities may be important for both in vitro Ames microbial assays and potential carcinogenic exposure in human therapy.