Presence Of Phenothiazines Research Articles

Pleiotropic actions of phenothiazine drugs are detrimental to Gram-negative bacterial persister cells

Bacterial persister cells are temporarily tolerant to bactericidal antibiotics but are not necessarily dormant and may exhibit physiological activities leading to cell damage. Based on the link between fluoroquinolone-mediated SOS responses and persister cell recovery, we screened chemicals that target fluoroquinolone persisters. Metabolic inhibitors (e.g., phenothiazines) combined with ofloxacin (OFX) perturbed persister levels in metabolically active cell populations. When metabolically stimulated, intrinsically tolerant stationary phase cells also became OFX-sensitive in the presence of phenothiazines. The effects of phenothiazines on cell metabolism and physiology are highly pleiotropic: at sublethal concentrations, phenothiazines reduce cellular metabolic, transcriptional, and translational activities; impair cell repair and recovery mechanisms; transiently perturb membrane integrity; and disrupt proton motive force by dissipating the proton concentration gradient across the cell membrane. Screening a subset of mutant strains lacking membrane-bound proteins revealed the pleiotropic effects of phenothiazines potentially rely on their ability to inhibit a wide range of critical metabolic proteins. Altogether, our study further highlights the complex roles of metabolism in persister cell formation, survival and recovery, and suggests metabolic inhibitors such as phenothiazines can be selectively detrimental to persister cells.

UV-Light Effects on Cytochrome C Modulated by the Aggregation State of Phenothiazines

The present study shows the factors that modulate the photodamage promoted by phenothiazines. Cytochrome c was irradiated with UV light for 120 min, over a pH range from 4.0 to 8.0, in the absence and in the presence of different concentrations of thioridazine (TR) and fluphenazine (FP). In the absence of phenothiazines, the maximal rate of a Soret band blue shift (nm/min) from 409 to 406 nm was obtained at pH 4.0 (0.028 nm/min). The presence of phenothiazines at the concentration range 10-25 µmol/L amplified and accelerated a cytochrome c blue shift (409 to 405 nm, at a rate = 0.041 nm/min). Above 25 µmol/L, crescent concentrations of phenothiazines contributed to cytochrome c protection with (maximal at 2500 µmol/L). Scanning electronic microscopy revealed the formation of nanostructures. The pH also influenced the effect of low phenothiazine concentrations on cytochrome c. Thus, the predominance of phenothiazine-promoted cytochrome c damage or protection depends on a balance of the following factors: the yield of photo-generated drug cation radicals, which is favored by acidic pH; the stability of the cation radicals, which is favored by the drug aggregation; and the cytochrome c structure, modulated by the pH.

RamA Confers Multidrug Resistance in Salmonella enterica via Increased Expression of acrB , Which Is Inhibited by Chlorpromazine

Salmonella enterica serovar Typhimurium SL1344, in which efflux pump genes (acrB, acrD, acrF, tolC) or regulatory genes thereof (marA, soxS, ramA) were inactivated, was grown in the presence of 240 antimicrobial and nonantimicrobial agents in the Biolog Phenotype MicroArray. Mutants lacking tolC, acrB, and ramA grew significantly worse than other mutants in the presence of 48 agents (some of which have not previously been identified as substrates of AcrAB-TolC) and particularly poorly in the presence of phenothiazines, which are human antipsychotics. MIC testing revealed that the phenothiazine chlorpromazine had antimicrobial activity and synergized with common antibiotics against different Salmonella serovars and SL1344. Chlorpromazine increased the intracellular accumulation of ethidium bromide, which was ablated in mutants lacking acrB, suggesting an interaction with AcrB. High-level but not low-level overexpression of ramA increased the expression of acrB; conferred resistance to chloramphenicol, tetracycline, nalidixic acid, and triclosan and organic solvent tolerance; and increased the amount of ethidium bromide accumulated. Chlorpromazine induced the modest overproduction of ramA but repressed acrB. These data suggest that phenothiazines are not efflux pump inhibitors but influence gene expression, including that of acrB, which confers the synergy with antimicrobials observed.

Stimulatory response to the oxidation of benzidine and other xenobiotics mediated by lipoxygenase in the presence of phenothiazines

To test the possibility that certain efficient substrates for lipoxygenase (LOX) produce shuttle oxidants that stimulate the generation of reactive oxygen species from other chemicals. Metabolic interactions were conducted in vitro between chlorpromazine (BZ) or other phenothiazines and benzidine or other xenobiotics mediated by soybean lipoxygenase (SLO) or purified human term placental lipoxygenase (HTPLO) in the presence of hydrogen peroxide, and the rates of xenobiotics oxidation were measured by spectroscopic analysis. Chlorpromazine cation radical (CPZ(*+)) generated by lipoxygenase triggered a rapid oxidation of benzidine to benzidine diimine cation. Under the experimental conditions used, the metabolic interaction resulted in a 42-fold greater stimulation than BZ alone in the rate of BZ oxidation. The magnitude of stimulation of benzidine oxidation exhibited a dependence on the pH of the reaction medium, amount of the enzyme, and concentration of chlorpromazine, BZ, and hydrogen peroxide. A number of other phenothiazines were also found to stimulate BZ oxidation, albeit to a lesser degree. Chlorpromazine cation radical stimulated the oxidation of all six other xenobiotics tested. The highest stimulation (94-fold) was noted in the oxidation of tetramethyl phenylenediamine to Wursters blue radical, while the lowest stimulatory response (2-fold) was observed in guaiacol. Preliminary data showed that purified HTPLO also displayed a similar stimulatory response to benzidine oxidation in the presence of chlorpromazine. Both soybean lipoxygenase and purified human term placental lipoxygenase can mediate stimulatory response to the oxidation of benzidine and other xenobiotics in the presence of phenothiazines.

DNA-Dependent Protein Kinase Is Inhibited by Trifluoperazine

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine nuclear kinase, important for the repair of DNA double strand breaks (DSB). Cells defective in DNA-PK show increased sensitivity to ionising radiation and different DNA-damaging drugs, such as cisplatinum. Increased sensitivity to cisplatinum has previously been noted in the presence of phenothiazines. We tested a panel of phenothiazines and one thioxanthen for any influence upon the activity and expression of DNA-PK in a nonsmall cell lung cancer cell line, U-1810. The activity of DNA-PK was completely inhibited in cell lysate and in purified enzyme by 200 μM TFP. DNA-PKcs and Ku86 cleavage were evident in U-1810 cells after 30 min incubation with 100 μM TFP, along with changes in the cells consistent with apoptosis. Our study suggests that phenothiazines and thioxanthens, acting through DNA-PK, have the potential to enhance the effects of DNA damaging agents.

Thermodynamics of partitioning and efflux of phenothiazines from liposomes.

The partitioning of nine phenothiazines between dimyristoylphosphatidylcholine (DMPC) liposomes and 0.9% wt/vol saline at pH 6 has been studied both below and above the phase transition temperature (Tc) of the phospholipid. Higher partitioning was observed above Tc. Both the entropy and enthalpy of partitioning were positive below and above Tc, and a linear relationship between the entropy and enthalpy has been derived. In general, the partitioning and transport of alkylaminophenothiazines in DMPC liposomes over the temperature range of 5 to 40 degrees C is entropically controlled. The entropies and enthalpies of partitioning of various groups in the phenothiazine structure have been calculated. No relationship was found between particle size of the DMPC liposomes and the equilibrium partition coefficient at 25 degrees C. However, the particle size of liposomes did increase with increasing acyl chain length of the phospholipid. Using differential scanning calorimetry, the enthalpy and entropy of transition of the DMPC liposomes in the absence and presence of phenothiazines has been calculated. The temperature dependence of the first-order rate constant of trimeprazine tartrate transport in DMPC liposomes was investigated and was found to be maximum at the Tc of the phospholipid.

Phosphorylated intermediates of CA 2+-ATPase and alkaline phosphatase in plasma membranes from rat duodenal epithelium

Purified basolateral membranes of rat duodenal epithelium were phosphorylated in the presence of [ γ- 32P]ATP and 1 μM free Ca 2+ under conditions favorable for phospho-intermediate formation. Analysis of 32P-labeled membrane proteins on SDS-acrylamide gel electrophoresis revealed the presence of three Ca 2+-sensitive phosphoproteins with molecular weights of 64 000 (protein I), 84 000 (II) and 115 000 (III). Both proteins I and II were identified as phospho-intermediate forms of alkaline phosphatase on the basis of the following criteria: these phosphoproteins were resistant to hydroxylamine and alkaline treatment, phosphate incorporation was stimulated by Zn 2+ but totally inhibited by excess β-glycerophosphate during phosphorylation. A similar labeling efficiency was obtained with inorganic 32P instead of [ γ- 32P]ATP. Protein III comigrated on SDS gels with the phosphorylated intermediate of Ca 2+-ATPase from rat heart sarcolemnal membranes and was identified as the intestinal form of Ca 2+-ATPase in view of the following observations: binding of 32P to this protein was hydroxylamine sensitive and alkaline labile, which is in agreement with an acylphosphate character of an ATPase intermediate, phospho-intermediate formation was dependent on low free Ca 2+ concentrations (1 μM) in the presence of 1 mM Mg 2+, phosphorylation was strongly inhibited in the presence of phenothiazines (0.1 mM chlorpromazine or trifluoperazine). The amount of 32P bound to Ca 2+-ATPase in basolateral membranes was only 6% of the amount incorporated into (Na + + K +)-ATPase. The turnover number of duodenal Ca 2+-ATPase estimated from this phosphorylation study is remarkably similar to the value for the red blood cell Ca 2+-ATPase. Phosphorylation experiments with purified leaky brush border membranes under similar conditions did not reveal a phospho-intermediate from of Ca 2+-ATPase as demonstrated in basolateral membranes. In contrast about 7-fold higher levels of 32P in phosphoprotein bands I and II were found. The 32P-labeling characteristics of these proteins were similar to those of alkaline phosphatase intermediates in the basolateral membrane. The results of this study are fully in support of an earlier study in which high affinity Ca 2+-ATPase was found exclusively in the basolateral membrane of duodenal epithelium.