Phototoxicity of phenothiazine derivatives. II. Photosensitized cross-linking of erythrocyte membrane proteins

Irradiation in the presence of O₂, with near-UV light of five promazine (PZ) derivatives added to erythrocyte ghost membranes, causes covalent cross-linking between proteins as revealed by a progressive decrease in the amounts of proteins separable by electrophoresis after denaturation. The induction of cross-links in the two spectrin subunits is a single-hit process as a function of the irradiation time; relatively the rate constants (in min^?1) of the photoreactions were 0.060 with chlorpromazine (CPZ), 0.039 with methoxypromazine (MTPZ), 0.031 with PZ, 0.029 with triflupromazine (TFPZ) and 0.006 with acepromazine (ACPZ).A main photochemical intermediate implicated in the spectrin aggregation seems to be the cation radical of the PZ derivatives. Indeed, (i) the chemically generated cation radicals can induce the reaction in the dark; (ii) the photoaggregation is regularly reduced upon addition of increasing concentrations of NaN₃; (iii) NaN₃ similarly affects the amount of cross-links induced by the isolated cation radicals. Hydroxyl radicals are also involved in the photocross-linking when the reaction is initiated only by MTPZ and not by the other sensitizers.In the absence of oxygen during irradiation, PZ, MTPZ and ACPZ completely loose their cross-linking activities whereas CPZ and TFPZ remain as efficient as in the presence of oxygen.     

吩噻嗪衍生物在体内外对肠道细菌的作用

本工作测试了７种吩噻嗪衍生物在体外对５株好氧或兼性厌氧菌和８９株厌氧菌的最小抑菌浓度,结果表明,吩噻嗪衍生物对细菌（尤其是球菌和厌氧菌）具有一定的抑制作用,但携带可拮抗艰难梭菌肠道定植屏障菌群的悉生小鼠接受２周或４周的Ｃｈｌｏｒｐｒｏｍａｚｉｎｅ（０．２ｍｇ／小鼠／天）并不会使菌群屏障遭破坏。     

Phenothiazine–rhodamine‐based colorimetric and fluorogenic ‘turn‐on' sensor for Zn2+ and bioimaging studies in live cells

A phenothiazine–rhodamine (PTRH) fluorescent dyad was synthesized and its ability to selectively sense Zn²⁺ ions in solution and in in vitro cell lines was tested using various techniques. When compared with other competing metal ions, the PTRH probe showed the high selectivity for Zn²⁺ ions that was supported by electronic and emission spectral analyses. The emission band at 528 nm for the PTRH probe indicated the ring closed form of PTRH, as for Zn²⁺ ion binding to PTRH, the λ_em get shift to 608 nm was accompanied by a pale yellow to pink colour (under visible light) and green to pinkish red fluorescence emission (under UV light) due to ring opening of the spirolactam moiety in the PTRH ligand. Spectral overlap of the donor emission band and the absorption band of the ring opened form of the acceptor moiety contributed towards the fluorescence resonance energy transfer ON mechanism for Zn²⁺ ion detection. The PTRH sensor had the lowest detection limit for Zn²⁺, found to be 2.89 × 10^?8 M. The sensor also demonstrated good sensing application with minimum toxicity for in vitro analyses using HeLa cells.     

New phenothiazine-type multidrug resistance modifiers: anti-MDR activity versus membrane perturbing potency

The phenothiazine multidrug resistance (MDR) modulators are chemically diversified but share the common feature to be hydrophobic cationic molecules. Molecular mechanisms of their action may involve interactions with either P-glycoprotein or membrane lipid matrix. In the present work we study the anti-MDR and biophysical membrane effects of new phenothiazine derivatives differing in the type of group substituting phenothiazine ring at position 2 (H-, Cl-, CF(3)-) and in the side chain group (NHCO(2)CH(3) or NHSO(2)CH(3)). Within each phenothiazine subset we found that anti-MDR activity (determined by P-glycoprotein inhibition assessed by flow cytometry) correlates with the theoretically calculated hydrophobicity value (logP) and experimental parameters (determined by calorimetry and fluorescence spectroscopy) of lipid bilayers. It is concluded that the biological and biophysical activity of phenothiazine derivatives depends more on the type of ring substitution than on the nature of the side chain group.     

Vitalfärbung mit Derivaten des Phenothiazins

Zusammenfassung Zellkulturen wurden vergleichend mit Handelsfarbstoffen und gereinigten Farbstoffen aus der Phenothiazingruppe (Thionin und seine methylierten Homologe — Azurfarbstoffe — bis zum Methylenblau vitalgefärbt.Granuläre Farbstoffspeicherung wird erst dann beobachtet, wenn im zweifach aminosubstituierten Phenothiazin (Thionin) mindestens zwei Methylgruppen gegen Protonen an den Aminogruppen ausgetauscht worden sind, wie dies bei Azur A der Fall ist.Dieser Befund läßt sich nur bei Verwendung gereinigter Farbstoffe erheben. Die hier verwendeten Handelsfarbstoffe Azur C sind in solchem Ausmaß mit höher methylierten Homologen vermischt, daß deren Effekt eine Eignung des Azur C zur granulären Farbstoffspeicherung vortäuscht. Gereinigtes Azur C ist dazu jedoch ungeeignet.Phasenoptisch erscheint die mit Azur A, Azur B und Methylenblau erreichte Farbstoffspeicherung mehr vakuolär als granulär. Die vom jeweils verwendeten Farbstoff induzierten vakuolären Einschlüsse zeigen morphologische Eigenheiten.Die Befunde werden diskutiert, unter besonderer Berücksichtigung der mit den kationischen Farbstoffen vermutlich reagierenden anionischen zellulären Bestandteilen.

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Vital staining with phenothiazine derivativesComparative application of commercial and purified dyes

Summary In a comparative study cells grown in monolayer have been vitally stained with commercial dye samples and dyes purified by thin layer chromatography and column chromatography. The dyes used were derivatives of amino-substituted phenothiazine (thionine to methylene blue).Granular storage of dyes has been observed only after the protons of the amino groups had at least been substituted by two methyl groups, as represented by the compound azure A.This observation is based on the use of purified dyes. Commercial samples of azure C are mixed with fractions of the higher methylated homologues of thionine to a degree that these impurities imply suitability of azure C for intracytoplasmic granular storage. Purified azure C, however, does not induce formation of cytoplasmic vacuoles.In the phase contrast microscope, inclusions formed under the influence of azure A, azure B and methylene blue exhibit a more vacuolar than granular appearance. There are morphologic differences, between vacuoles formed under the influence of the respective dyes.The observations are discussed with special regard to the anionic cellular constituents supposed to react with the cationic dyes.

Die Arbeit wurde mit Unterstützung der F. Hoffmann-La Roche und Co. AG., Basel, durchgeführt.     

Single-Electron-Transfer in the Reduction of 1, 2-Dioxetanes by Biologically Active Substrates

Substances of low oxidation potential, which can also make available protons and hydrogen atoms, e.g. phenothiazines. NADH, and ascorbic acid efficiently reduce 1, 2-dioxetanes to their vic-diols by single-electron-transfer; a significant side reaction is catalytic decomposition of dioxetanes into the corresponding ketone fragments     

Phototoxicity of phenothiazine derivatives. I. Inactivating and mutagenic effects on bacteriophage φX174

Inactivation of φX174 bacteriophages as a function of the irradiation time in the near-UV and in the presence of triflupromazine (TFPZ), promazine (PZ), chlorpromazine (CPZ) or methoxypromazine (MTPZ) proceeds according to single hit kinetics. Acepromazine (ACPZ) has no significant activity. At low concentrations (0.1 mM) TFPZ and PZ are the most active compounds. Higher concentrations (up to 5 mM) result in a protective effect by these two compounds but cause increased inactivation rates in the case of MTPZ or CPZ. Photoinactivation mediated by TFPZ or CPZ increases the reversion frequency of a φXamber mutant. Neither MTPZ nor PZ sensitization induces mutagenesis. The effect of NaN₃ on the phage inactivation rate varies depending upon both the sensitizer and the concentration of the quencher. Phage inactivation in an N₂ atmosphere is measurable only in the presence of high concentrations of CPZ and MTPZ. The drugs do not show any selectivity for calf thymus DNA or bovine serum albumin, at least as measured by dialysis equilibrium experiments.     

Inhibition of axonal transport in vitro in frog sciatic nerves by chlorpromazine and lidocaine

Summary The effects of chlorpromazine hydrochloride (CPZ HCl) and prochlorperazin-metansulfonate (PCPZ) on the fast axonal transport of labelled proteins were examined in vitro in a peripheral frog nerve.A 0.1 mM concentration of CPZ HCl and PCPZ reduced the amount of transported proteins by more than 50 per cent. An almost complete block was obtained with a 0.5 mM concentration of these two drugs. The lower concentration hardly affected the protein synthesis. The transport inhibiting effect of 0.1 mM of the drugs was reversible but not that of the higher concentration.The number of microtubuli was strongly decreased and the number of filaments increased at the transport inhibiting concentrations. The ultrastructural changes induced by 0.1 mM of the phenothiazine tranquilizer were largely reversible. The local anesthetics lidocaine (18.3 mM) and tetracaine (3.3 mM) both caused similar changes, i.e. a reduction in the number of microtubuli. No ultrastructural effects were observed after treatment with 1 mM ouabain. These three drugs are known to block the axonal flow in the present system at the above mentioned concentrations.The biochemical and ultrastructural results are discussed in relation to those induced by other drugs affecting axonal transport.The present work was supported by grants from Statens Naturvetenskapliga Forskningsråd (No. 2535-8), C.-B. Nathorsts Vetenskapliga och Allmännyttiga Stiftelser, the Swedish Medical Research Council (B73-12X-2543-05B), H. Hierta's Stiftelse and W. och M. Lundgrens Stiftelse. Thanks are due to Mrs B. Egnér, Mrs E. Fjällstedt, Mrs. E. Norström and Mrs U. Svedin for expert technical assistance.     

Pro‐oxidant effects of phenothiazine and phenoxazine on erythrocytes in the presence of peroxyl radical

Phenothiazine (PtzNH) and phenoxazine (PozNH) can protect human erythrocytes against hemolysis induced by 2,2′‐azobis(2‐amidinopropane hydrochloride) (AAPH), a peroxyl radical supplier. However, an antioxidant may be a pro‐oxidant to accelerate the oxidation in the presence of radicals. The aim of this work is to assess whether PtzNH and PozNH have the potential to be pro‐oxidants in AAPH‐induced hemolysis of human erythrocytes. It has been found that high concentrations of PtzNH and PozNH employed were able to initiate hemolysis even in the absence of AAPH. In the presence of AAPH, the period of PtzNH and PozNH to lag hemolysis (t_lag) decreased with the increase in the concentrations of PtzNH and PozNH, implicating that high concentration of PtzNH and PozNH accelerated hemolysis. So, PtzNH and PozNH played pro‐oxidants' role in this case. Furthermore, high concentrations of AAPH employed made the pro‐oxidant effect of PtzNH more remarkable. On the contrary, PozNH played a pro‐oxidant role if only low concentration of AAPH was employed. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:280–286, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20290     

Hollow fiber-liquid phase microextraction combined with gas chromatography for the determination of phenothiazine drugs in urine

A simple method of hollow fiber-liquid phase microextraction (HF-LPME) combined with gas chromatography (GC) was developed for the analysis of four phenothiazine drugs (promethazine, promazine, chlorpromazine and trifluoperazine) in human urine samples. All variables affecting the extraction of target analytes including organic solvent type, stirring rate, extraction time, extraction temperature, pH of sample solution and ionic strength were carefully studied and optimized. Under the optimal conditions, the analytical performance of HF-LPME-GC-flame photometric detector (FPD) and HF-LPME-GC-flame ionization detector (FID) were evaluated and compared. The results showed that the HF-LPME-GC-FID was more sensitive than HF-LPME-GC-FPD for the determination of four target phenothiazine drugs, while the signal peak shape and resolution obtained by HF-LPME-GC-FPD was better than that obtained by HF-LPME-GC-FID. HF-LPME-GC-FPD/FID was successfully applied for the assay of the interested phenothiazine drugs in urine sample, and the excretion of the drugs was also investigated by monitoring the variation of the concentration of chlorpromazine in urine of a psychopath within 8 h after drug-taking. The proposed method provided an effective and fast way for the therapeutic drug monitoring (TDM) of phenothiazine.