Effect of gamma-linolenic acid and prostaglandins E1 on gamma-radiation and chemical-induced genetic damage to the bone marrow cells of mice

The effect of gamma-linolenic acid (GLA) and prostaglandin E1 (PGE1) on gamma-radiation, diphenylhydantoin (DPH), benzo(a)pyrene (BP), and 4-alpha-phorbol-induced genetic damage to the bone marrow cells of mice, using the sensitive micronucleus (MN) test was investigated. PGE1 and its precursor GLA prevented gamma-radiation, DPH, BP, and 4-alpha-phorbol-induced genetic damage.     

Diphenylhexatriene as a Fluorescent Probe for Monitoring Low Density Lipoprotein Peroxidation

The use of the fluorescent probe diphenylhexatriene (DPH) for monitoring low density lipoprotein (LDL) peroxidation has been investigated. The DPH incorporation into LDL results in a high fluorescence signal which decreases with time after addition of cupric ions. A strong correlation was found between the decay of the DPH fluorescence signal and the appearance of the thiobarbituric reactive substances (TBARS). HPLC and spectrofluorometric analyses demonstrated that DPH is destroyed during the time course of the copper-induced LDL peroxidation. The decrease in DPH fluorescent signal is prevented by addition of EDTA, vitamin E and drugs which protect LDL against peroxidation such as probucol or calcium antagonists. The high fluorescence of DPH allows the use of very small quantities of LDL (less than 5 μg/ml LDL protein). We thus suggest that DPH could be of use for continuous monitoring of LDL autooxidation, especially for the in vitro testing of the protective effect of antioxidant compounds.     

High-content imaging of neutral lipid droplets with 1,6-diphenylhexatriene

Neutral lipid droplets (LDs) are dynamic lipid storage organelles found in all eukaryotic cells from yeast to mammals and higher plants. LDs are important to many physiological processes that include basic cellular maintenance, metabolism, and diverse medical pathologies. LD accumulation has been studied extensively by a range of methods, but particularly by microscopy with several fluorescent dyes extensively used for qualitative and quantitative imaging. Here, we compared established LD stains Nile Red and BODIPY 493/503 to the 4', 6-diamidino-2-phenylindole (DAPI)-range dye 1,6-diphenyl-1,3,5-hexatriene (DPH; excitation/emission λmax=350 nm/420 nm) using high-content image analysis. HeLa cells treated with oleic acid or vehicle were used to compare staining patterns between DPH and Nile Red as well as DPH and the LD protein adipophilin. DPH, Nile Red, and BODIPY 493/503 were compared as assay reagents in oleic acid dose-response experiments. Treatment of MCF-7 cells with sodium butyrate was used as a second cellular system for high-content analysis of LD formation. In this experimental context, we demonstrate the compatibility of DPH with GFP, a technical limitation of Nile Red and BODIPY 493/503 dyes. These data show that DPH has comparable sensitivity and specificity to that of Nile Red. Z'-factor analysis of dose-response experiments indicated that DPH and BODIPY 493/503 are well suited for quantitative analysis of LDs for high-throughput screening (HTS) applications.     

Short-lived fluorescence component of DPH reports on lipid--water interface of biological membranes

Fluorescence measurements of 1,6-diphenyl-1,3,5-hexatriene (DPH) in large unilamellar phospholipid vesicles were performed to characterize the influence of the membrane physical properties on the short-lived lifetime component of the fluorescence decay. We have found that the short-lived component of DPH significantly shortens when the membrane undergoes a temperature-induced phase transition as it is known for the long-lived component of DPH. We induced membrane phase transitions also by alcohols, which are reported to be distributed different way in the membrane–ethanol close to the membrane-water interface and benzyl alcohol in the membrane core. A different effect of the respective alcohol on the short and long decay component was observed. Both the time-resolved fluorescence spectra of DPH taken during lipid vesicle staining and the lifetime dependences caused by changes of temperature and/or induced by the alcohols show that the short-lived fluorescence originates from the population of dye molecules distributed at the membrane–water interface.     

Temperature, pressure and cholesterol effects on bilayer fluidity; a comparison of pyrene excimer/monomer ratios with the steady-state fluorescence polarization of diphenylhexatriene in liposomes and microsomes

Pyrene excimer/monomer (E/M) ratios have been compared with the steady-state fluorescence polarization (P) of diphenylhexatriene (DPH) in multilamellar liposomes of dilaurylphosphatidylcholine and rat liver microsomes. The purpose was to use the well-understood properties of DPH to reveal the nature of bilayer fluidity which pyrene manifests as an E/M ratio. Reducing the temperature (from 37 degrees C to 8 degrees C), increasing the hydrostatic pressure (from 0.1 to 70 MPa), and, in liposomes, cholesterol enrichment (up to 0.30 mole fraction) separately decreased the E/M ratios and increased P. The pyrene membrane/buffer partition coefficient was affected by temperature but not by pressure, and in the case of cholesterol enrichment, it was assumed to be unaffected. Plots of P as a function of the E/M ratio showed the two to be closely correlated (r = 0.99 in liposomes and 0.96 in microsomes), independent of the treatment used to reduce fluidity. The apparent activation volume and enthalpy for excimer formation was calculated and compared with published data. Pyrene E/M ratios probably reflect the intermolecular volume (fluidity) of the outer region of the bilayer, which is reduced by a decrease in temperature and an increase in pressure and cholesterol. DPH reports the bilayer interior, which is similarly ordered by the experimental treatments. The regional distinction between the two probes, however, accounts for the divergence of E/M ratios and P, which has been reported in membranes enriched with fluidizing fatty acids.     

The phenyltetraene lysophospholipid analog PTE-ET-18-OMe as a fluorescent anisotropy probe of liquid ordered membrane domains (lipid rafts) and ceramide-rich membrane domains

The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15'-phenylpentadeca-8',10',12',14'-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine) is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333-5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997-10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.     

Effects of diphenylhydantoin and phenobarbital on voltage-clamped myelinated nerve.

Diphenylhydantoin (DPH) and phenobarbital (PB) have a selective action in blocking spontaneous activity in nerves made hyperexcitable by lowering the calcium concentration of the bathing medium (Rosenberg, P. and Bartels, E. 1967 J. Pharmacol. Exp. Ther. 155, 532-544.). To investigate this further, we examined the action of DPH and PB on voltage-clamped single myelinated nerves at two different calcium concentrations. In 1.8 mM calcium Ringer, DPH reduced the sodium permeability (PNa) without affecting the potassium conductance (GK) or the voltage-dependent time constants of sodium activation (taum) and inactivation (tauh), and potassium activation (taun). PB was similar to DPH except that in addition to reducing PNa, it shifted taum in the direction of depolarization. When the calcium concentration was lowered to 0.36 mM, the curves relating taum and taun to membrane potential were shifted in the direction of hyperpolarization, as expected. However, the addition of DPH or PB reduced or abolished these shifts. It is suggested that both DPH and PB stabilize hyperexcitable membranes by an action on the parameter m, and that this may contribute to their antiepileptic action.     

Interaction of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene with biomembranes

The relationship between the conditions of membrane labelling by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and its fluorescence parameters was investigated. In the labelling solutions prepared by the usual method, the presence of DPH microcrystals was revealed which led to the lower resultant fluorescence anisotropy values. Lower labelling efficiency was observed with DPH solutions in tetrahydrofuran when compared with solutions in acetone. Modifications of the labelling procedure are proposed which give better reproducibility of the results. There modified method involves the preparation of a 2 X 10(-4) mol. 1(-1) DPH stock solution in acetone, a 100-fold dilution in an appropriate buffer, subsequent bubbling through with nitrogen for 30 min and mixing the resulting solution with cell/membrane suspension in a 1:1 (v/v) ratio. Changes in intensity, anisotropy and spectra of DPH fluorescence in the course of membrane labelling were studied. A two-stage model of the incorporation of DPH into membranes was proposed, according to which DPH molecules first quickly adhere to the membrane surface and then are slowly translocated to the apolar regions of the membrane.     

Voltage-dependent modulation of TRPA1 currents by diphenhydramine

Diphenhydramine (DPH) has been broadly used to treat allergy. When used as a topical medicine, DPH temporarily relieves itching and pain. Although transient receptor potential type A1 (TRPA1) channel is known to play roles in both acute and chronic itch and pain, whether DPH affects the activities of TRPA1 remains unclear. Using whole-cell patch clamp recordings, we demonstrated that DPH modulates the voltage-dependence of TRPA1. When co-applied with a TRPA1 agonist, DPH significantly enhanced the inward currents while suppressing the outward currents of TRPA1, converting the channel from outwardly rectifying to inwardly rectifying. This effect of DPH occurred no matter TRPA1 was activated by an electrophilic or non-electrophilic agonist and for both mouse and human TRPA1. The modulation of TRPA1 by DPH was maintained in the L906C mutant, which by itself also causes inward rectification of TRPA1, indicating that additional acting sites are present for the modulation of TRPA1 currents by DPH. Our recordings also revealed that DPH partially blocked capsaicin evoked TRPV1 currents. These data suggest that DPH may exert its therapeutic effects on itch and pain, through modulation of TRPA1 in a voltage-dependent fashion.     

Deviation from homeoviscous adaptation in Escherichia coli membranes.

The process by which an organism changes the composition of its membranal fatty acids in response to growth temperature, so as to maintain optimal membrane functioning, is known as homeoviscous adaptation (HA). One expression of HA is the constancy of the fluorescence polarization (P) of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in membranes of cells grown at various temperatures. The P of DPH in the membranes of Escherichia coli was shown by us to be inversely proportional to bacterial growth rate on different carbon sources. This result, implying failure of HA, is now complemented by measurements of DPH lifetimes, which indicate that the dominant variables contributing to the drop in P are (a) the order parameter of the membrane, which goes down, and (b) the fluidity, which may slightly increase. These are then the changes induced by enhanced growth rate. Two additional effects, cell membrane permeability and sensitivity to thermal shock, determined by the diffusion of o-nitrophenylgalactoside (ONPG) and by exposure to 52 degrees C, respectively, are reported to increase with growth rate. We can now conclude that there is a deviation from the principle of HA in E. coli grown at various rates, brought about by controlling the growth media at constant temperatures.     

Effects of some biologically active compounds on phagosome-lysosome fusion in peritoneal macrophages of mice.

Effects of biologically active compounds bilirubin (BR), farmorubicin (FR), and chelerythrine (CR) on phagosome-lysome (P-L) fusion in mouse peritoneal macrophages were studied using fluorescent dye acridine orange as lysosomal labelling and yeast cells as target. It was found that all three compounds tested enhanced P-L fusion. To investigate mechanisms of these effects, changes in fluidity of rat liver lysosomal membranes under influence of BR, FR and CR were studied by measuring fluorescence intensity, lifetime, and polarization of DPH or TMA-DPH incorporated in isolated rat liver lysosomes. In order to characterize the cytoskeleton changes under the action of these biologically active compounds F-actin content in peritoneal macrophages of mice was determined. Our results demonstrate that BR action induces a decrease in DPH and TMA-DPH polarization, FR increases DPH and TMA-DPH polarization, and CR causes only an increase in TMA-DPH polarization in lysosomal membranes. All three compounds tested increase F-actin content in peritoneal macrophages. Thus, the effect of BR on P-L fusion is connected with increasing fluidity of lysosomal membranes and the cytoskeleton changes. The enhancement of P-L fusion under the action of FR and CR can most likely be explained by changes of the cytoskeleton state.     

Molecular dynamics simulation of a dye molecule in the interior of a bilayer: 1,6-diphenyl-1,3,5-hexatriene in dipalmitoylphosphatidylcholine

A molecular dynamics simulation was carried out for a dipalmitoylphosphatidylcholine (DPPC) membrane in its liquid crystalline state containing different concentrations of the dye molecule 1,6-diphenyl-1,3,5-hexatriene (DPH). From a numerical analysis of the trajectories, we obtained information concerning structural changes of the membrane due to the presence of the probe and some hydrodynamic information concerning the probe itself. The hydrodynamic properties regarding dye molecules that have been reported in this article are: rotational and translational diffusion coefficient and relaxation times. From this analysis, we estimated a range of values of 0.6-0.9 cP for the micro-viscosity in the mid-membrane. These simulations also afforded us some information regarding structural changes in the membrane as a consequence of the presence of the fluorescent dyes at different concentrations. Thus, the disorder inside the membrane, the surface area per lipid and thickness of the membrane were also investigated.     

The effect of parahydroxylation of diphenylhydantoin on metaphase accumulation.

DPH has a colchicine-like action on metaphase arrest of cultured human lymphocytes. The first step in detoxification of DPH increased its power to accumulate metaphases 3-fold. This hydroxy derivative [5-[4-hydroxyphenyl]-5-phenylhydantoin, HPPH] 3.6 X 10(-4) M was equivalent colchicine 1 x 10(-5) M in its power to inhibit metaphase completion. The effect of HPPH on mitosis was reversible; colchicine effect was not reversed and vincristine effect was partially reversed by washing drug from the medium. Hydroxylation of DPH did not change its inhibition of DNA synthesis and enhanced inhibition of protein synthesis to a minor degree. Detoxification increased the colchicine-like action of DPH.     

The effects of cholesterol on the time-resolved emission anisotropy of 12-(9-anthroyloxy)stearic acid in dipalmitoylphosphatidylcholine bilayers

The time-resolved fluorescence emission anisotropy of 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) have been measured in dipalmitoylphosphatidylcholine liposomes in the presence and absence of 40 mol% cholesterol at temperatures above and below the phase transition temperature (41°C). By using a synchronously-pumped mode-locked frequency-doubled dye laser and single photon counting detection with an excitation response function of 300 picosecond, rotational correlation times down to less than 1 nanosecond could be resolved. Whereas DPH showed only small changes in the limiting anisotropy on the addition of cholesterol, 12-AS showed significant increases in this parameter with the effect being potentiated at higher temperatures. This difference in behaviour has been attributed to a fluorophore-cholesterol interaction that resulted in a change in the fluorophore geometry. Not only do DPH and 12-AS sense different depolarizing rotations due to the different directions of their emission dipoles but also differ in their lipid interactions which alter their limiting anisotropies. The implication is that the comparison of steady-state anisotropy measurements between chemically identical fluorophores in different lipid environments may be complicated by molecular distortions that change the motions to which the steady-state fluorescence parameters will be sensitive.     

Effect of chronic administration of phenytoin on regional monoamine levels in rat brain

Phenytoin (DPH) is a widely used anticonvulsant drug but a conclusive mode of action is not yet clear. This study was undertaken to assess the effects of chronic administration of DPH on monoamine levels. DPH (50 mg/kg body weight) was administered to adult male Wistar rats by intraperitoneal injections for 45 days and the regional brain levels of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) were assayed using high performance liquid chromatographic (HPLC) method. The experimental rats revealed no behavioral deficits of any kind nor body and brain weight deficits were observed. Increased NE levels were observed after DPH administration in motor cortex (P<0.05), striatum-accumbens (P<0.01) and hippocampus (P<0.01), whereas, NE level was decreased in brain stem (P<0.05). DA levels were increased in striatum-accumbens (P<0.05), hypothalamus (P<0.001) and cerebellum (P<0.001) but decreased in brainstem (P<0.01). In DPH treated rats, 5-HT levels were increased in motor cortex (P<0.001) but decreased in cerebellum (P<0.001) when compared to control group of rats. The present study suggest that chronic administration of DPH induces alterations in monoamine levels in specific brain regions. DPH seems to mediate, its anticonvulsant action by selectively altering the monoamine levels in different brain regions.     

Lack of mutagenicity of diphenylhydantoin in in vitro short-term tests

The mutagenicity of diphenylhydantoin (DPH) and its major metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), has been re-evaluated by the Ames test using Salmonella typhimurium and, for DPH only, by an in vitro cytogenetic test with human lymphocytes and a turbidimetric assay of tubulin polymerization. As negative results were obtained in all test systems used here, one has to conclude that DPH is devoid of mutagenic properties.     

Nicotinic acetylcholine receptor induces lateral segregation of phosphatidic acid and phosphatidylcholine in reconstituted membranes

Purified nicotinic acetylcholine receptor (AChR) protein was reconstituted into synthetic lipid membranes having known effects on receptor function in the presence and absence of cholesterol (Chol). The phase behavior of a lipid system (DPPC/DOPC) possessing a known lipid phase profile and favoring nonfunctional, desensitized AChR was compared with that of a lipid system (POPA/POPC) containing the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the AChR. Fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and AChR intrinsic fluorescence by a nitroxide spin-labeled phospholipid showed that the AChR diminishes the degree of DPH quenching and promotes DPPC lateral segregation into an ordered lipid domain, an effect that was potentiated by Chol. Fluorescence anisotropy of the probe DPH increased in the presence of AChR or Chol and also made apparent shifts to higher values in the transition temperature of the lipid system in the presence of Chol and/or AChR. The values were highest when both Chol and AChR were present, further reinforcing the view that their effect on lipid segregation is additive. These results can be accounted for by the increase in the size of quencher-free, ordered lipid domains induced by AChR and/or Chol. Pyrene phosphatidylcholine (PyPC) excimer (E) formation was strongly reduced owing to the restricted diffusion of the probe induced by the AChR protein. The analysis of Forster energy transfer (FRET) from the protein to DPH further indicates that AChR partitions preferentially into these ordered lipid microdomains, enriched in saturated lipid (DPPC or POPA), which segregate from liquid phase-enriched DOPC or POPC domains. Taken together, the results suggest that the AChR organizes its immediate microenvironment in the form of microdomains with higher lateral packing density and rigidity. The relative size of such microdomains depends not only on the phospholipid polar headgroup and fatty acyl chain saturation but also on AChR protein-lipid interactions. Additional evidence suggests a possible competition between Chol and POPA for the same binding sites on the AChR protein.     

Human papillomavirus type 16 E5 protein affects cell-cell communication in an epithelial cell line.

The human papillomavirus type 16 (HPV16) E5 protein is considered to have weak oncogenic properties, and its function in infected human keratinocytes is unknown. HPV16 E5 protein has been found to localize to the Golgi apparatus and the plasma membrane. To analyze the effect of E5 on plasma membrane properties, cells from the human keratinocyte cell line HaCaT were transfected with the HPV16 E5 open reading frame under the control of an inducible promoter. The gap junction-mediated cell-cell communication of E5- and vector-transfected cells was analyzed by microinjection of Lucifer yellow to measure dye coupling of the cells. A strong impairment of dye transfer in E5-transfected cells but not in vector-transfected cells was observed, with more than 80% dye transfer inhibition 40 min after injection. This impairment correlated with dephosphorylation of connexin 43, the major gap junctional protein in HaCaT cells. Furthermore, the dye coupling inhibition was not the result of differentiation of the E5-expressing cells, since no overexpression of cytokeratin 1 or filaggrin, markers of HaCaT cell differentiation, could be observed. These results therefore strongly suggest a correlation between expression of the HPV16 E5 open reading frame, impairment of gap junction-mediated dye coupling, and dephosphorylation of connexin 43.     

Correlation of beta-amyloid aggregate size and hydrophobicity with decreased bilayer fluidity of model membranes

beta-amyloid peptide (Abeta) is the primary constituent of senile plaques, a defining feature of Alzheimer's disease. Aggregated Abeta is toxic to neurons, but the mechanism of toxicity remains unproven. One proposal is that Abeta toxicity results from relatively nonspecific Abeta-membrane interactions. We hypothesized that Abeta perturbs membrane structure as a function of the aggregation state of Abeta. Toward exploring this hypothesis, Abeta aggregate size and hydrophobicity were characterized using dynamic and static light scattering and 1,1-bis(4-anilino)naphthalene-5,5-disulfonic acid (bis-ANS) fluorescence. The effect of Abeta aggregation state on the membrane fluidity of unilamellar liposomes was assessed by monitoring the anisotropy of the membrane-embedded fluorescent dye, 1,6-diphenyl-1,3,5-hexatriene (DPH). Unaggregated Abeta at pH 7 did not bind bis-ANS and had little to no effect on membrane fluidity. More significantly, Abeta aggregated at pH 6 or 7 decreased membrane fluidity in a time- and dose-dependent manner. Aggregation rate and surface hydrophobicity were considerably greater for Abeta aggregated at pH 6 than at neutral pH and were strongly correlated with the extent of decrease in membrane fluidity. Prolonged (7 days) Abeta aggregation resulted in a return to near-baseline levels in both bis-ANS fluorescence and DPH anisotropy at pH 7 but not at pH 6. The addition of gangliosides to the liposomes significantly increased the DPH anisotropy response. Hence, self-association of Abeta monomers into aggregates exposes hydrophobic sites and induces a decrease in membrane fluidity. Abeta aggregate-induced changes in membrane physical properties may have deleterious consequences on cellular functioning.     

A direct method for the correction of pressure-induced scrambling of polarized fluorescence intensities.

A simple and direct method for the simultaneous correction of steady-state polarized fluorescence intensities, depolarized (or scrambled) by the effects of applied hydrostatic pressure, is described. In the method discussed here, it is not necessary to first determine the scrambling factors from a separate experiment with a dye immobilized in a rigid medium. Rather correction for depolarizing effects of the high-pressure spectroscopy cell windows is achieved by direct recalculation of the measured polarized data obtained for the sample of interest at the time of data collection. This method of correction is tested for common fluorescent dyes 1, 6-diphenyl-1,3,5-hexatriene (DPH) and 9,10-diphenylanthracene in glycerol where their rotational behavior is well understood. In addition, the pressure-induced "melt" profile for the more complicated biologically relevant system of DPH imbedded within dipalmitoylphosphatidylcholine small unilamellar vesicles has been reexamined. While the method discussed here is used for the correction of steady-state polarized data, it may be easily adapted for use in time-resolved polarized fluorescence measurements. Advantages and limitations of the new correction method are discussed.