Dihydrorhodamine 123 is superior to 2,7-dichlorodihydrofluorescein diacetate and dihydrorhodamine 6G in detecting intracellular hydrogen peroxide in tumor cells

Dihydrorhodamine 123 (DHR 123), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and dihydrorhodamine 6G (DHR 6G) were evaluated as probes for detecting cellular hydrogen peroxide levels in SPC-A-1 lung adenocarcinoma cells. Imaging techniques and fluorescence-activated cell scan were used in the study of the probe responses. Obvious green fluorescence was established after a 25-min exposure. After staining with MitoTracker Orange CM-H2TMRos (a probe for mitochondria) and the abovementioned probes simultaneously, only the DHR 123 and DHR 6G groups exhibited legible green fluorescence in the mitochondrial regions. Furthermore, the DHR 6G group exhibited weaker fluorescence intensity. When 100 microM H2O2 was added to SPC-A-1 cells loaded with these probes, the intracellular fluorescence increased rapidly and significantly. Our results suggest that DHR 123 is superior for the instantaneous detection of cellular hydrogen peroxide in SPC-A-1 cells.     

Implications of using the fluorescent probes, dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate, for the detection of UVA-induced reactive oxygen species

During investigation of UVA-induced oxidative stress in HaCaT keratinocytes with dihydrorhodamine 123 (DHR123) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), exaggerated baseline values were observed within control samples, suggesting a mechanism of probe oxidation and subsequent change in fluorescence intensity (FI) independent of cellular ROS generation. The effects of diluent, UVA pre-treatment and loading protocols upon the FI of the probes have therefore been investigated. The study confirmed the capacity of Dulbecco's Modified Eagle's Medium (DMEM) to confer fluorescence intensity changes in both probes, most notably DCF-DA. In addition, UVA pre-treatment compromises the effectiveness of DHR123 and DCF-DA to detect ROS generated in a cell-free system. In vitro data shows a greater UVA-induced FI increase in HaCaT cells loaded with probe before rather than after UVA treatment. This study has important implications for future research, the understanding of previous studies and associated confounding effects using DHR123 and DCF-DA as ROS sensitive probes.     

Active oxygen chemistry within the liposomal bilayer. Part IV: Locating 2',7'-dichlorofluorescein (DCF), 2',7'-dichlorodihydrofluorescein (DCFH) and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) in the lipid bilayer

2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) is commonly used to detect the generation of reactive oxygen intermediates and for assessing the overall oxidative stress in toxicological phenomenon. It has been suggested that DCFH-DA crosses the cell membrane, subsequently undergoing deacetylation by intracellular esterases. The resulting 2',7'-dichlorodihydrofluorescein (DCFH) is proposed to react with intracellular hydrogen peroxide or other oxidizing ROS to give the fluorescent 2',7'-dichlorofluorescein (DCF). Using an NMR chemical shift-polarity correlation, we have determined that DCFH-DA and DCFH are located well within the lipid bilayer and certainly not at the interface. These results, therefore, put into serious question the proposed ability of DCFH to come in contact with the aqueous phase and thereby interact with aqueous intracellular ROS and components. However, H2O2 and superoxide can cross or at least penetrate the lipid bilayer and react with certain lipophilic substrates. This may well describe the mode of reaction of these and other ROS with DCFH.     

Reactivity of 2',7'-dichlorodihydrofluorescein and dihydrorhodamine 123 and their oxidized forms toward carbonate, nitrogen dioxide, and hydroxyl radicals

The aim of this study was to investigate the oxidation of two common fluorescent probes, dichlorodihydrofluorescein (DCFH2) and dihydrorhodamine (DHR), and their oxidized forms, dichlorofluorescein and rhodamine, by the radical products of peroxynitrite chemistry, *OH, NO2*, and CO3*-. At pH 8.0-8.2, rate constants for the interaction of carbonate radical with probes were estimated to be 2.6 x 10(8) x M(-1) s(-1) for DCFH2 and 6.7 x 10(8) M(-1) s(-1) for DHR. Nitrogen dioxide interacted more slowly than carbonate radical with these probes: the rate constant for the interaction between NO2* and DCFH2 was estimated as 1.3 x 10(7) M(-1) s(-1). Oxidation of DHR by nitrogen dioxide led to the production of rhodamine, but the kinetics of these reactions were complex. Hydroxyl radical interacted with both probes with rate constants close to the diffusion-controlled limit. We also found that oxidized forms of these fluorescent probes reacted rapidly with carbonate, nitrogen dioxide, and hydroxyl radicals. These data suggest that probe oxidation may often be in competition with reaction of the radicals with cellular antioxidants.     

Comparison of spectrum-shifting intracellular pH probes 5'(and 6')-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7, 1'(3'H)-isobenzofuran]-3'-one and 2',7'-biscarboxyethyl-5(and 6)-carboxyfluorescein.

The dyes carboxy-SNARF-1 and BCECF are fluorescent probes of intracellular pH that exhibit changes in spectral shape upon proton binding which allow one to use measurements of fluorescence at two or more wavelengths in order to measure pH without artifacts associated with variability in dye loading, etc. In evaluating these dyes for this study, whole spectra, rather than measurements at two wavelengths, were analyzed. For BCECF, the effects of the intracellular milieu were minimal: both the pH-sensitive excitation spectrum and the pKa agreed closely with values found in extracellular solution. In contrast, both the spectra and the pKa for the emission spectrum-shifting carboxy-SNARF-1 showed significant differences between intracellular and extracellular dye. As a result, extremely misleading values for intracellular pH will be obtained if one attempts to use extracellular dye to calibrate intracellular carboxy-SNARF-1 measurements. Multiple origins were found for the discrepancy: (i) the intracellular dye was found to be significantly quenched, with the deprotonated form being more strongly quenched than the protonated form; and (ii) the pKa for the equilibrium with intracellular hydrogen ions was shifted by +0.2 pH units. These effects were readily reversed by disruption of the cell, but were not due to sequestering of dye in an acidic cell compartment.     

Characterization of 5(6)-carboxy-2,'7'-dichlorofluorescein transport by MRP2 and utilization of this substrate as a fluorescent surrogate for LTC4

MRP2 (ABCC2) is an efflux transporter expressed on the apical membrane of polarized cells. This protein has a major role in the biliary elimination of toxic compounds from the liver. As MRP2 transports many endogenous compounds, including LTC4 as well as xenobiotics and toxic phase II metabolites, blockade of this transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. The vesicular transport assay is a great tool to study drug-drug and drug-endogenous compound interactions of ABC transporters. In this assay, inside-out membrane vesicles are used, so the test compound can readily access the transporter. As MRP2 transports many ionic compounds that are difficult to investigate in a whole-cell system because of permeability reasons, the vesicular transport assay is a good choice for screening MRP2-mediated interactions. LTC4 is not an optimal substrate for high-throughput screening for MRP2 interactors, even though it is an important MRP2 substrate. Therefore, the transport of a drug surrogate, 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), by MRP2 was characterized using the vesicular transport assay. The data indicate that CDCF proves to be an ideal substrate for MRP2 vesicular transport assay with its optimal detection and transport properties.     

Non-covalent interaction of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein with proteins and its application

The interactions of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein (TBTCF) with BSA, ovalbumin (OVA) and poly-L-lysine (PLYS) at pH 3.70 have been investigated by combination of the spectral correction technique and the Langmuir isothermal adsorption. The active connection actions such as ion pairs, van der Waals' force, hydrogen bond, hydrophobic bond were proposed to explain the non-covalent interaction between TBTCF and BSA, OVA and PLYS. Effects of the electrolyte and high temperature indicated that union of the active connections between TBTCF and BSA and OVA was too firm to be destroyed. The relationship between the binding number of TBTCF and variety fraction of the amino acid residues was analyzed. The binding number of TBTCF depended on the number of positively charged amino acid residues. The other amino acid residues surrounded and seized TBTCF by hydrogen bonds and hydrophobic bonds when the electrostatic attraction pulled TBTCF to link protein. In addition, a novel method named the absorbance ratio difference was established for determination of protein in trace level and was applied with much higher sensitivity than the ordinary method.     

Detection of human autoantibodies specific for 5'-m7GMP and m7G(5')ppp(5')N

An enzyme-linked immunosorbent assay was utilized for the detection of spontaneously occurring antibodies with apparent specificities for m7G, 5'-m7GMP, and m7G(5')ppp(5')C. From the sera of 50 patients containing anti-nuclear antibodies, 48 (96%) possessed antibodies which bound to one or more immobilized nucleoside-BSA antigens (A-, G-, C-, U-, and T-BSA). Additionally, 8 (16%) of these sera contained immunoglobulins that reacted with m7G-BSA antigen. In these latter sera, soluble competitors such as m7G, 5'm7GMP, and m7G(5')ppp(5')C (but not 5'-AMP, -GMP, -CMP, -UMP, and -TMP or m1G and m22G) effectively inhibited antibody-binding to immobilized m7G-BSA. These results indicate the existence of spontaneously occurring anti-m7G antibodies in autoimmune diseases which are distinct from anti-G antibody populations.     

Simultaneous quantification of oxidative stress and cell spreading using 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein.

BACKGROUND: Mitochondrial dysfunction may lead to increased oxidative stress and consequent changes in cell spreading. Here, we describe and validate a novel method for simultaneous quantification of these two parameters. METHODS: Human skin fibroblasts were loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-H(2)DCF), and its oxidative conversion into CM-DCF was monitored as a function of time by video-rate confocal microscopy and real-time image averaging. Cell size was determined after binarization of the acquired images. RESULTS: At the lowest practical laser output, CM-DCF formation occurred with zero order kinetics, indicating that [CM-H(2)DCF] was not rate-limiting and that the rate of [CM-DCF] formation (V(CM-DCF)) was a function of the cellular oxidant level. Analysis of fibroblasts of a healthy control subject and a patient with a deficiency of NADH:ubiquinone oxidoreductase, the first complex of the oxidative phosphorylation system, revealed a significant increase in cellular oxidant level in the latter cells that was, however, not accompanied by a change in cell spreading. Conversely, chronic treatment with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a derivative of vitamin E, markedly decreased the oxidant level and cell spreading in both control and patient fibroblasts. CONCLUSIONS: We present a reliable method for simultaneous quantification of oxidant levels and cell spreading in living cells.     

Synthesis and antitumor activity of duocarmycin derivatives: A-ring pyrrole compounds bearing beta-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group

A series of A-ring pyrrole derivatives of duocarmycin bearing beta-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group were synthesized, and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. New Seg-B analogues bearing beta-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group containing double bond as spacer had lower peripheral blood toxicity than the derivatives bearing 5',6',7'-trimethoxyindole-2'-carboxyl group in Seg-B of the natural type. Moreover, most of them exhibited potent antitumor activity against in vivo murine tumor models.     

2' (or 3')-O-(2, 4, 6-trinitrophenyl)adenosine 5'-triphosphate as a probe for the binding site of heavy meromyosin ATPase.

1. From NMR, IR and visible absorption studies of 2'(or 3')-O-(2, 4, 6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP), 2'(or 3')-O-(2, 4, 6-trinitrophenyl) adenosine (TNP-Ad(, and 1-(2'-hydroxyethoxy)-2, 4, 6-trinitrobenzene (TNP-EG), it was concluded that there is an intramolecular interaction between the base and 2, 4, 6-trinitrophenyl (TNP) moieties in the TNP-ATP molecule. 2. A broad new absorption band was observed in the 530-630 nm region when excess indole was added to reaction mixtures containing TNP-ATP dissolved in 50% methanol or dimethyl sulfoxide. On addition of aromatic amino acid derivatives, methanol or dimethyl sulfoxide. On addition of aromatic amino acid derivatives, TNP-ATP and TNP-Ad underwent spectral shifts in the 400-550 nm region. The formation of a 1:1 complex apparently occurred between TNP-ATP and aromatic amino acid derivatives, and the complex with N-acetyltryptophan was stable in 50% methanol. The difference spectrum of TNP-EG vs. TNP-ATP closely resembled that induced by the addition of N-acetyltryptophan to the TNP-ATP solution. 3. The binding of 2'(or 3')-O-(2, 4, 6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) to heavy meromyosin (HMM) was studied by the rapid gel equilibrium method using Sephadex G-25. A dissociation constant of 1.4 muM and a maximum binding number of 1.8 were obtained in 0.15 M KCl, 10 mM MgCl2, and 50 mM Tris-HCl (pH 8.0) at 25 degrees. TNP-ADP bound to the enzyme caused a characteristic spectral shift in the visible region. This spectral shift was explained in terms of an interaction between tryptophanyl residues and the adenine base of TNP-ADP bound to the enzyme. TNP-ADP quenched the tryptophanyl fluorescence, but TNP-EG and TNP-Ad did not. In the presence of 6 M guanidine hydrochloride, TNP-ADP scarcely quenched the tryptophanyl fluorescence, its effect being comparable to that of TNP-Ad.     

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 5: Exploration of pyridazinones containing 6-amino-substituents

The synthesis of 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones bearing 6-amino substituents as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC(50)<0.10 microM). In vitro DMPK data (microsome t(1/2), Caco-2 P(app)) for many of the compounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the NS5B protein is discussed.     

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 3: Further optimization of the 2-, 6-, and 7'-substituents and initial pharmacokinetic assessments

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. Lead optimization led to the discovery of compound 3a, which displayed potent inhibitory activities in biochemical and replicon assays [IC(50) (1b)<10nM; IC(50) (1a)=22 nM; EC(50) (1b)=5nM], good stability toward human liver microsomes (HLM t(1/2)>60 min), and high ratios of liver to plasma concentrations 12h after a single oral administration to rats.     

Synthesis and antiviral activity assay of novel (E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine

(E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

Synthesis and stability studies of 2',3',5'-tri-O-acetyl-2-amino(-N6-cyclopentyl)-1-deazaadenosines

In this article, we report on the synthesis of 2',3',5'-tri-O-acetyl-2-amino-1-deazaadenosine and of 2',3',5'-tri-O-acetyl-2-amino-N(6)-cyclopentyl-1-deazaadenosine, which are very versatile intermediates for the preparation of 2-substituted 1-deazaadenosine derivatives. The two synthesized compounds showed to be quite unstable, with the N(6)-substituted derivatives being less stable than the N(6)-unsubstituted counterpart, according to the calculated HOMO-LUMO energy gap. Stability studies were performed through HPLC-MS analysis.