Mechanism of inhibition of porcine leukocyte 12-lipoxygenase by the isoform-specific inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid

The mechanism of inhibition of porcine leukocyte 12-lipoxygenase by 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) was investigated. This compound is selective for the leukocyte form of the 12-lipoxygenase and inhibits the purified recombinant enzyme with an IC(50) value of approximately 2 microM. OPP induced a concentration-dependent lag phase in the oxygenation of arachidonic acid and decreased the maximal rate of reaction. Addition of the fatty acid hydroperoxide 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE) to the reaction greatly reduced the OPP-induced lag. Lineweaver-Burk analysis of the effect of OPP on 12-lipoxygenase kinetics with arachidonic acid indicated that it was a mixed-type inhibitor. OPP was not metabolized by 12-lipoxygenase as evidenced by its quantitative recovery from incubations with stoichiometric amounts of enzyme and 13-HPODE or arachidonic acid. OPP inhibited the pseudoperoxidase activity of the enzyme with 13-HPODE and the reducing agent, BWA137C. Lineweaver-Burk analysis of the effect of OPP on pseudoperoxidase kinetics suggested that OPP was competitive with 13-HPODE. Single-turnover experiments indicated that OPP inhibited the reduction of 13-HPODE by a stoichiometric amount of ferrous 12-lipoxygenase. Addition of 13-HPODE shortened the OPP-induced lag phase but did not affect the maximal rate of enzyme activity. In addition, OPP had no effect on total product formation in either the presence or the absence of 5 microM 13-HPODE when the reaction was allowed to go to completion. All of these observations are consistent with a model for inhibition of 12-lipoxygenase activity in which OPP slows the oxidation of the inactive ferrous enzyme to the active ferric enzyme and competes with arachidonic acid for the ferric enzyme.     

Induction of chromosome aberrations and sister-chromatid exchanges in CHO-K1 cells by o-phenylphenol

o-Phenylphenol (OPP), is used in Japan as a fungicide in food additives for citrus fruits. The induction of chromosome aberrations and sister-chromatid exchanges (SCEs) by OPP in cultured Chinese hamster ovary (CHO-K1) cells was studied. Cells were exposed to various concentrations of OPP ranging from 50 to 175 micrograms/ml for 3 h, and further incubated for 27 and 42 h. These incubation periods are almost equal to 2 and 3 cell cycles. SCEs and chromosome aberrations were induced by OPP at concentrations of 100, 125 and 150 micrograms/ml after the incubation for 27 h. For chromosome aberrations, chromatid breaks and exchanges there was a dose-dependent increase. Diplochromosomes due to endoreduplication were also caused by the same concentrations of OPP in a dose-dependent manner. After incubation for 42 h, chromosome aberrations were also increased by OPP at concentrations of 100 and 125 micrograms/ml, but the frequencies of SCEs were not significantly different from those of the control. These results suggest that OPP has a cytogenetic toxicity, and that the DNA damage resulting in SCEs induced by OPP is relatively short-lived and can be repaired during the longer incubation time.     

Ames test-negative carcinogen, ortho-phenyl phenol, binds tubulin and causes aneuploidy in budding yeast

Ortho-phenyl phenol (OPP) is broad-spectrum of fungicides and antibacterial agents. OPP tested negative in an Ames system and positive with respect to the formation of tumors in the urinary bladder in rats when administered in diet, showing attributes of an Ames test-negative carcinogen. It has also been demonstrated that OPP does not bind or cleave DNA in vivo or in vitro, rather dose-dependent protein binding in OPP-treated rats was observed. OPP, however, generates chromosomal aberrations including aneuploidy. Thus, the steps by which Ames test-negative carcinogens exert their effects need to be elucidated. Here, we used an assay of loss of heterozygosity (LOH) in Saccharomyces cerevisiae to determine the biological effects of OPP and its hepatic metabolite phenyl hydroquinone (PHQ). LOH was found to be induced by OPP and PHQ because of a functional chromosome loss: aneuploidy. PHQ bound to and interfered with the depolymerization of tubulin in vitro and arrested the cell-cycle at M and G1. These results indicate that OPP and PHQ damaged tubulin to cause mis-segregation of chromosome by delaying cell-cycle progression through mitosis, and as a consequence caused aneuploidy.     

Impact of Apoplast Volume on Ionic Relations in Plant Cells

This study was performed to investigate the effect of cholesterol content, surface charge and sterical stabilization on the physico-chemical properties of liposomes prepared from the cancerostatic alkylphospholipid, octadecyl-1,1-dimethyl-piperidino-4-yl-phosphate (D21266), and their relationship to in vitro cytotoxicity. Stable incorporation of OPP into liposomes was found to be highly dependent on the cholesterol content. ³¹P-NMR spectroscopy as well as analysis of the lipid composition of OPP-containing liposome formulations revealed an increase in the amount of non-liposome-associated, micellar OPP as the cholesterol content decreased. The fraction of non-liposome-associated OPP constituted about 10% of total OPP when cholesterol was present in equimolar amounts (45.5/45.5 mol %) and increased to approximately 30% at a twofold excess of OPP over cholesterol (58.8/29.4 mol %). In monolayer incorporation studies it was shown that the existence of an increasing micellar pool of lipids leads to increased lipid transfer into the target monolayer. Liposome formulations containing more OPP than cholesterol were also found to display greater cytotoxicity. However, all liposome formulations were less cytotxic than pure (micellar) OPP. Cytotoxicity was not affected by the incorporation of N-methoxy-polyethyleneglycol₂₀₀₀-phosphoethanolamine, a lipid that is known to reduce liposome uptake into phagocytic cells. The results demonstrate that the increase in cell toxicity correlates with the increase in non-liposome-associated, micellar OPP, which can readily exchange into cellular membranes. Received: 4 October 2000/Revised: 29 March 2001     

Sister-chromatid exchanges and cell-cycle delay in Chinese hamster V79 cells treated with 9 organophosphorus compounds (8 pesticides and 1 defoliant)

Significant increase of sister-chromatid exchanges (SCE) in V79 cells treated with 2 organophosphorus pesticides (OPP), fenthion and oxydemeton-methyl, was observed. The other 7 compounds (6 OPP and 1 defoliant) namely, amaze, azinphos-methyl, bolstar, DEF-defoliant, fensulfothion, monitor and nemacur caused no increase of SCE frequencies at the doses tested. All the compounds except fensulfothion and oxydemeton-methyl induced cell-cycle delay in varying degrees. Cell-cycle delay caused by an OPP was found to be dose-dependent. Based on these data as well as others reported, it would appear that OPP which induce no SCE increase and no or slight cell-cycle delay could be considered as good candidates to substitute the pesticides that have been found to be harmful to the environment.     

Improving Sprint Performance in Soccer: Effectiveness of Jump Squat and Olympic Push Press Exercises

Training at the optimum power load (OPL) is an effective way to improve neuromuscular abilities of highly trained athletes. The purpose of this study was to test the effects of training using the jump squat (JS) or Olympic push-press (OPP) exercises at the OPL during a short-term preseason on speed-power related abilities in high-level under-20 soccer players. The players were divided into two training groups: JS group (JSG) and OPP group (OPPG). Both groups undertook 12 power-oriented sessions, using solely JS or OPP exercises. Pre- and post-6 weeks of training, athletes performed squat jump (SJ), countermovement jump (CMJ), sprinting speed (5, 10, 20 and 30 m), change of direction (COD) and speed tests. To calculate the transfer effect coefficient (TEC) between JS and MPP OPP and the speed in 5, 10, 20, and 30 m, the ratio between the result gain (effect size [ES]) in the untrained exercise and result gain in the trained exercise was calculated. Magnitude based inference and ES were used to test the meaningful effects. The TEC between JS and VEL 5, 10, 20, and 30 m ranged from 0.77 to 1.29, while the only TEC which could be calculated between OPP and VEL 5 was rather low (0.2). In addition, the training effects of JS on jumping and speed related abilities were superior (ES ranging from small to large) to those caused by OPP (trivial ES). To conclude, the JS exercise is superior to the OPP for improving speed-power abilities in elite young soccer players.     

Interaction of alkylphospholipid liposomes with MT-3 breast-cancer cells depends critically on cholesterol concentration

We have investigated interaction of alkyphospholipid (APL) liposomes consisting of 1,1-dimethylpiperidin-1-ium-4-yl) octadecyl phosphate (OPP) and different concentrations of cholesterol (CH) with human MT-3 breast-cancer cells using electron paramagnetic resonance method (EPR) with advanced characterization of EPR spectra of spin labeled liposome membranes. After incubation of OPP liposomes with MT-3 cells, a reduction of liposome entrapped, water soluble spin-probe tempocholine (ASL) was observed, indicating that ASL is released from liposomes and is reduced by oxy-redoxy systems inside the cells. This process is fast if cholesterol content in the bilayer was 29 or 45 mol%, whereas at 56 mol% cholesterol the process is almost stopped. The rate of spin-probe reduction in first 10 min after incubation with cells is even faster as for the free ASL, indicating that liposomes with low amount of cholesterol accelerate penetration of ASL into the cells. A faster release of hydrophilic material from liposomes with low cholesterol content coincides with the presence of domains with highly disordered alkyl chain motion that disappears at 50 mol% of cholesterol. We propose that these highly fluid domains are responsible for interaction of OPP liposomes with cells and fast release of the entrapped material into the cells. These results suggest that micelles are not the only reason for cytotoxic effect of OPP liposome formulations, as it was suggested before. OPP in liposomes, containing 45 mol% cholesterol or less, also contributes to the cytotoxic effect, due to their fast interaction with breast-cancer cells.     

In vivo genotoxicity of ortho-phenylphenol, biphenyl, and thiabendazole detected in multiple mouse organs by the alkaline single cell gel electrophoresis assay

In Japan, ortho-phenylphenol (OPP), biphenyl (BP), and thiabendazole (2-(4'-thiazolyl)benzimidazole, TBZ) are commonly used as a postharvest treatment to preserve imported citrus fruits during transport and storage. We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of those agents in mouse stomach, liver, kidney, bladder, lung, brain, and bone marrow. CD-1 male mice were sacrificed 3, 8, and 24 h after oral administration of the test compounds. OPP (2000 mg/kg) induced DNA damage in the stomach, liver, kidney, bladder, and lung, BP (2000 mg/kg) and TBZ (200 mg/kg) induced DNA damage in all the organs studied. For OPP, increased DNA damage peaked at 3–8 h and tended to decrease at 24 h. For BP, on the contrary, increased DNA migration peaked at 24 h. That delay may have been due to the fact that OPP is metabolized by cytochrome 450 and prostaglandin H synthase to phenylbenzoquinone (PBQ), a DNA binding metabolite, and BP is metabolized to PBQ via OPP and m-phenylphenol. The positive response to TBZ, an aneugen, supports the in vivo DNA-damaging action of TBZ.     

High consistency enzymatic hydrolysis of hardwood substrates

The feasibility of using a laboratory peg mixer to carry out high consistency enzymatic hydrolysis of lignocellulosic substrates was investigated. Two hardwood substrates, unbleached hardwood pulp (UBHW) and organosolv pretreated poplar (OPP), were used in this study. Hydrolysis of UBHW and OPP at 20% substrate consistency led to a high glucose concentration in the final hydrolysate. For example, a 48 h enzymatic hydrolysis of OPP resulted in a hydrolysate with 158 g/L of glucose. This is the highest glucose concentration ever obtained from enzymatic hydrolysis of lignocellulosic substrates. Fermentation of UBHW and OPP hydrolysates with high glucose content led to high ethanol concentrations, 50.4 and 63.1 g/L, respectively after fermentation. Our results demonstrate that using common pulping equipment to carry out high consistency hydrolysis can overcome the rheological problems and greatly increase the sugar and ethanol concentrations after the hydrolysis and fermentation.     

Genotoxic effects of o-phenylphenol metabolites in CHO-K1 cells

The effects of microsomal activation and/or deactivation on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) in cultured Chinese hamster ovary cells (CHO-K1 cells) by o-phenylphenol (OPP) were studied, and concurrently the metabolites were determined. After a 3-h incubation in the presence of 15% S9 mix (45 microliters/ml of S9), OPP (25-150 micrograms/ml) dose-independent SCEs and chromosomal aberrations were induced, while the amount of phenylhydroquinone (PHQ) metabolite produced from OPP did not increase linearly in the higher doses. The maximum induction of chromosomal aberrations was 18% at the 150 micrograms/ml dose, and of SCEs 13.8/cell at 75 micrograms/ml. The corresponding control values were 3% and 5.8/cell. The lowest dose required to induce SCEs in the presence of S9 mix was 25 micrograms/ml. Changing the percent of S9 mix (0-50%) while holding the OPP dose constant (100 micrograms/ml) produced a correlation between SCEs and the production of PHQ. PHQ caused cytogenetic effects both with and without S9 mix, however, in the absence of S9 mix it was more lethal and was oxidized to phenylbenzoquinone (PBQ). These results suggest that the enhanced cytogenetic effects of OPP by the addition of S9 mix correlated with the amount of PHQ produced or with the further oxides of PHQ such as phenylsemiquinone and/or PBQ which are capable of being produced from PHQ spontaneously or by the mixed-function oxidase system.     

Glucose metabolism in proliferating epithelial cells from the rat colon.

1. The effects of fasting and fasting followed by refeeding on the activities of the oxidative pentose pathway (OPP) and the tricarboxylic acid cycle (TCA) in isolated rat colonocytes were estimated by the rate of production of 14CO2 from [1-14C]glucose and [6-14C]glucose, respectively. 2. Refeeding after a fast induced a 2-3-fold increase in glucose flux through the OPP and TCA cycle and the degree of change was similar in colonocytes from the proximal and distal colon. 3. Butyrate at a concentration of 40 mM inhibited the OPP by 20-30% (P less than 0.05) but had no effect on the activity of the TCA cycle. Glutamine at a concentration of 2 mM decreased the glucose flux through both the OPP and the TCA cycle by 30-50% (P less than 0.05). 4. Production of 14CO2 from the oxidation of butyrate or glucose indicated that the former was 5-7 times more active in colonocytes from fasted rats. After refeeding, however, butyrate utilization was similar to fasting values in the proximal colon but significantly lower (P less than 0.05) in the distal colon.     

New PCR based markers allowed to identify <Emphasis Type="Italic">Secale</Emphasis> chromatin in wheat-<Emphasis Type="Italic">Secale africanum</Emphasis> introgression lines

The genus of Secale has many agronomically important characters. In order to use the best of this species, markers tracking the rye chromatin incorporated into wheat must be developed. In this study, one rye genome-specific random amplified polymorphic DNA (RAPD) marker was isolated from Secale africanum (R^a genome). Two cloned markers, named OPP13₁₁₆₅ and OPP13₆₆₂, were 1165 bp and 662 bp, respectively. Sequence analysis revealed that OPP13₁₁₆₅ was highly homologous to a part of a new class of transposon-like gene called the Revolver family, and OPP13₆₆₂ was partially similar to LTR gypsy-like retrotransposon. Fluorescence in situ hybridization (FISH) showed only OPP13₁₁₆₅ localized within the whole arms of rye except their terminal regions and no signal was detected on wheat chromosomes, while OPP13₆₆₂ had no hybridization signal detected on wheat and rye genomes. Based on these sequences, two pairs of sequence-characterized amplified region (SCAR) primers were designed, and the resulted SCAR markers were able to target both cultivated and wild Secale species. The FISH patterns and the two SCAR markers should be able to identify and track all wheat-rye translocation lines, especially the S. africanum chromatin.     

Sulfhydryl compounds inhibit the cyto- and geno-toxicity of o-phenylphenol metabolites in CHO-K1 cells

The effects of cysteine and reduced glutathione (GSH) on the genotoxicity of o-phenylphenol (OPP) and its metabolites, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), were examined using the frequency of sister-chromatid exchanges (SCEs) and chromosome aberrations in CHO-K1 cells as parameters. Cytotoxic (cell-progression delay) and cytogenetic effects induced by a 3-h treatment with OPP, PHQ (100 micrograms/ml) or PBQ (50 micrograms/ml) with S9 mix after a 27-h expression time were inhibited by cysteine or GSH (3-10 mM). Materials corresponding to the cysteine or GSH adducts were found by HPLC in each incubation mixture. In the culture without S9 mix, PHQ and PBQ showed severe cytotoxicity since no metaphases could be obtained at doses over 25 and 5 micrograms/ml, respectively, and the sulfhydryl compounds inhibited the toxicity by the formation of adducts with PBQ and by inhibiting the formation of PBQ in the case of PHQ. With PHQ, the sulfhydryl compounds appeared to inhibit autooxidation. However, the sulfhydryl compounds did not inhibit the cytotoxic and cytogenetic effects caused by OPP in the cell mixture without S9 mix, but on the contrary intensified them. No adduct formation was detected in the incubation solution. On the basis of these results, it is considered that electrophilic quinone (PBQ) and/or semiquinone (phenylsemiquinone, PSQ) radicals, capable of binding to nucleophilic small molecules (such as cysteine and GSH) or (biological) macromolecules, are produced from metabolite PHQ in metabolic oxidation of OPP, and induce cyto- and geno-toxic effects in the cells. The cyto- and geno-toxic effects of OPP itself to the cells are clearly independent of any electrophilic radical reaction.     

Synthesis and characterization of chromium(III) octaphenylporphyrin complexes with various axial ligands: An insight into porphyrin distortion

Influence of axial ligands, MeCN, H₂O, py, and piperidine (pip), on distortion of (2,3,5,10,12,13,15,20-octaphenylporphinato)chromium(III), [Cr(OPP)]⁺, was investigated by X-ray crystallography and UV-vis and ESR spectroscopies. In crystal structures of [Cr(OPP)(MeCN)(H₂O)]ClO₄ · MeCN and [Cr(OPP)(H₂O)₂]ClO₄ · 3THF, the OPP²⁻ ligand had a planar structure. On the other hand, crystal structures of [Cr(OPP)(pip)₂]ClO₄ · 2CH₂Cl₂ and [Cr(OPP)(py)₂]ClO₄ exhibited a waved structure and a saddle-shaped structure with ruffling, respectively. In the UV-vis spectrum of [Cr(OPP)(py)₂]ClO₄ in CH₂Cl₂, the large red shift of the Soret band was observed. Furthermore, the small D value of 0.10 cm⁻¹ was obtained for [Cr(OPP)(py)₂]ClO₄ from the ESR spectrum in frozen 1,2-dichloroethane solution at 5 K. These results suggest that the OPP²⁻ ligand is distorted both in solid and in solution, and that the axial ligand would exert some effects on the porphyrin distortion.     

Synthesis, characterization, and distortion properties of vanadyl complexes of octaphenylporphyrin and dodecaphenylporphyrin

In order to investigate the influence of ligand distortion on metal centers of porphyrin complexes, distorted vanadyl porphyrin complexes, VO(OPP) (OPP = 2,3,5,10,12,13,15,20-octaphenylporphinato) and VO(DPP) (DPP = 2,3,5,7,8,10,12,13,15,17,18,20-dodecaphenylporphinato), have been prepared. In the crystal structures, VO(OPP) and VO(DPP) had a ruffled structure and a saddle-shaped structure, respectively. In addition, these complexes exhibited red shift and broadening of the absorption bands in the UV-Vis spectra and significant negative shifts of oxidation potentials of the porphyrin ligands in the cyclic voltammograms compared with those of the planar VO(TPP) (TPP = tetraphenylporphinato). These results indicate that the OPP and DPP complexes have the distorted structures both in solids and in solutions. The VO bond characters of VO(TPP), VO(OPP), and VO(DPP) do not show the significant difference in their crystal structures and resonance Raman spectra. This suggests that the distortion of porphyrin ligand little affects the Lewis acidity of the metal center. The non-planar porphyrin distortion gives the change of HOMO-LUMO gap.     

Application of the Melle-Boinot process to the fermentation of xylose by Pachysolen tannophilus

Pectinase production by Tubercularia vulgaris using orange-pulp pellets (OPP) or citrus pectin as carbohydrate sources was investigated. The highest levels of extracellular polygalacturonase were detected with OPP as inducing substrate. The pectinases present in the crude filtrate from T. vulgaris cultures included methyl esterase, endo-polygalacturonase and exo-polygalacturonase activities with both substrates. Moreover, in medium supplemented with OPP, the additional presence of one endo-polygalacturonase and two exo-polygalacturonases was detected.     

DNA cleavage by phenylhydroquinone: the major metabolite of a fungicide o-phenylphenol

The interaction of o-phenylphenol (OPP) and its metabolites with DNA was examined. As a model system, the reactivities of OPP and its metabolites with DNA were studied by using pUC18 DNA. The major metabolite formed in vitro from OPP by mixed function oxidase was phenylhydroquinone (PHQ). This result corresponds to the findings that PHQ in the form of glucuronide conjugate was the main product detected in bladder of OPP fed rats in vivo. When supercoiled pUC18 DNA (form I) was incubated with PHQ at concentrations from 1 X 10(-6) M to 2 X 10(-1) M, DNA strand scission by PHQ was observed at a concentration as low as 1 X 10(-5) M and the amount of linear form (form III) increased with increasing PHQ concentration. PHQ causes DNA strand scission. The DNA cleavage by OPP and phenyl-p-benzoquinone (PBQ) was barely detectable. The DNA cleavage by PHQ was inhibited by superoxide dismutase (SOD), catalase and several oxygen radical scavengers such as polyethylene glycol, tert-butanol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine. The production of superoxide radical from PHQ was confirmed by cytochrome c reduction assay. These results indicate that the oxygen radicals such as superoxide, hydroxyl radicals and some others generated in the process of oxidation of PHQ in aqueous solution are responsible for the DNA cleavage. In order to identify the sites of cleavage of DNA by PHQ, a 5'-end 32P-labeled 206 base-pair EcoRI-BglI fragment of pUC18 DNA was incubated with PHQ. The DNA was then analyzed by sequencing gel electrophoresis followed by autoradiography. When the DNA was incubated with PHQ and further treated with piperidine, cleavage was detected relatively more frequently at guanine residues. The attack seemed to occur at guanine residues in general, but was not restricted to guanines with specific residues in the vicinity.     

Molecular identification of five Indian sciaenids (pisces: perciformes,sciaenidae) using RAPD markers

The randomly amplified polymorphic DNA (RAPD) markers were used to detect interspecific genetic variability and genetic relatedness among five Indian sciaenids namely Otolithes cuvieri, Johnieops sina, Johnieops macrorhynus, Johnieops vogleri and Protonibea diacanthus for the first time. Eight RAPD primers (OPA01, OPA06, OPA07, OPA18, OPP12, OPP14, OPP16 and OPP11) generated 40 species specific diagnostic bands. The highest genetic divergence was detected between J. macrorhynus and P. diacanthus (0.586) where as the lowest one was observed between J. sina and J. vogleri (0.274). Handling editor: C. Strumbauer     

Depigmenting action of phenylhydroquinone,an O-phenylphenol metabolite,on the skin of JY-4 black guinea-pigs

The effects of o-phenylphenol (OPP) and its metabolite, phenylhydroquinone (PHQ) on the skin of JY-4 black guinea-pigs were studied. Topical application of 1 or 5% PHQ on the black skin of the back caused marked depigmentation and hypopigmentation of the skin after 5 weeks, whereas OPP applied at the same concentrations had little effect. Depigmented skin had an increased L* (lightness) value in the CIE-L*a*b* color system. This corresponded with a decreased number of melanocytes and melanosomes in the melanocytes and keratinocytes, the disruption of melanosomes in the melanocytes, and destruction of the membranous organelles of the melanocytes. These morphological and numerical changes in epidermal melanocytes indicate that selective melanocyte toxicity occurred. Furthermore, application of PHQ to the skin of white guinea-pigs caused skin irritation, as shown by a colorimetric increase in a* value (redness) and by histological observation of inflammation. This study confirmed that OPP, which is a reported depigmenter, has little depigmenting action, while its metabolite, PHQ, is a potent depigmenter preferentially affecting melanocytes.     

The influence of cholesterol and charge on the membrane domains of alkylphospholipid liposomes as studied by EPR

Alkylphospholipids are physiologically active derivatives of lipids effective in the treatment of breast cancer. Among them, octadecyl-(1,1-dimethyl-4-piperidino-4-yl)-phosphate (OPP) was demonstrated recently to have the strongest antitumor effect in micellar as well as in sterically stabilised liposome suspension with a low cholesterol content. In this work electron paramagnetic resonance (EPR) was used to study the influence of cholesterol, charge, and sterical stabilisation by PEG2000DSPE on the domain structure and fluidity characteristics of the membrane of OPP liposomes. As a spin probe 5-doxylpalmitoyl methyl ester was used. By computer simulation of the EPR spectra it was found that the experimental spectra are composed of three spectral components, which were attributed to three types of domains with different fluidity characteristics. The EPR parameters as well as the proportions of the individual domains were found to be mainly dependent on the amount of cholesterol, and only to a minor degree on charge and sterical stabilisation. There was a pronounced increase in the proportion of membrane domains with low order parameter, when the molar ratio of cholesterol to OPP was decreased below 1. At the same time the order parameters of all domains decreased, pointing to a transition from a less to a more fluid membrane organisation. These results coincide with an improved therapeutic activity of formulations with a low molar ratio of cholesterol to OPP and indicates that the fluidity characteristics of the membrane may be important for the effectiveness of liposomal alkylphospholipids against breast cancer cells.