A new method to purify hepatic CYP1A of Prochilodus scrofa, a Brazilian freshwater fish.

Cytochromes P450 constitute a superfamily of the phase I enzymes whose primary task is the detoxification of both endogenous and xenobiotic compounds. Fish, among non-mammalian species, have received great interest because they are a direct food source for humans as well as conveyors of toxic chemicals to human beings. The aim of the present study was the purification of the hepatic isoform of CYP1A in Prochilodus scrofa (Prochilodontidae), a Brazilian fish, using only one chromatographic step. The purification of CYP1A was done by Reverse Phase HPLC on a C18 column. Purified CYP1A was characterized with respect to electrophoretic, immunochemical and biocatalyst properties. CYP1A fractions produced a single uniform band on SDS-PAGE with an apparent molecular mass of 58 kDa. Purified CYP1A of P. scrofa showed strong cross-reactivity with antibodies directed against CYP1A from trout. The fraction was also encapsulated in two different reconstituted systems; one composed of neutral lipids and another of negatively charged lipids. In both of them, we could detect EROD activity but not PROD activity, which confirms that the CYP1A was purified with all its enzyme activity. There was an increase of activity when CYP1A and NADPH cytochrome P450 (CYP) reductase were encapsulated in negatively charged lipids, which confirms that the charge of lipid is essential to CYP1A activity. All these characteristics strongly suggest that this new procedure is efficient for purifying hepatic CYP1A from P. scrofa, showing that the CYP1A isoform of this fish has a highly conserved protein region.

Solubilization of human erythrocyte membranes by non-ionic surfactants of the polyoxyethylene alkyl ethers series.

In the present study, we investigated the interaction of the non-ionic surfactants polyoxyethylene alkyl ethers (C(n)E(m)) with erythrocyte membranes. For this purpose we have performed hemolytic assays under isosmotic conditions with five surfactants in the 8 polyoxyethylene ether series. By applying to the hemolytic curves a quantitative treatment developed for the study of surface-active compounds on biomembranes, we could calculate the surfactant/lipid molar ratios for the onset of hemolysis (R(e)(sat)) and for complete hemolysis (R(e)(sol)). This approach also allowed the calculation of the binding constants for each surfactant to the erythrocyte membrane. Results in the C(n)E(m) series were compared to those obtained for Triton X-100, a well-known non-ionic surfactant with values of cmc and HLB in the range of the alkyl ethers studied. Inside the series the lytic effect increased with the more hydrophobic homologues (C(10)E(8)

Effect of bilirubin on toxicity induced by trifluoperazine, dibucaine and praziquantel to erythrocytes.

Unconjugated bilirubin (UCB), like trifluoperazine (TFP), dibucaine (DBC) and praziquantel (PZQ), induces erythrocyte morphological changes, lysis and lipid exfoliation. In the present study we determined whether TFP, DBC and PZQ toxicity to erythrocytes was potentiated or reverted by UCB. Human erythrocytes were either treated or non-treated with 34.2 micromol/L UCB for 10 min prior to the incubation with toxic concentrations of TFP (0.12 mmol/L), DBC (1.5 mmol/L) or PZQ (3.0 mmol/L), for 1 h (37 degrees C). Studies of toxic effects included morphological analysis of erythrocytes, evaluation of hemoglobin release and loss of membrane lipids. Although UCB has an echinocytogenic effect, its co-incubation with TFP or PZQ did not alter the stomatocytogenic effect of the drug but enhanced DBC-induced stomatocytosis. Cell fusion was a common feature in experiments with DBC. Injurious effect of DBC to erythrocytes was potentiated by UCB as manifested by a marked increase in hemolysis (171%, p<0.05), and in elution of membrane cholesterol (73%, p<0.01) and phospholipids (123%, p<0.01). In opposite, toxic events produced by TFP and PZQ to erythrocytes were not aggravated by UCB. Interestingly, UCB prevented the loss of membrane cholesterol by PZQ (-36%, p<0.01), as well as that of phospholipids by TFP (-28%, p<0.05). These findings indicate that UCB potentiates DBC injury to erythrocytes, while protects membrane lipid elution by PZQ and TFP. Therefore, the relation of the benefits and risks of the administration of DBC to jaundiced patients should be carefully considered.

The liver monooxygenase system of Brazilian freshwater fish.

Content of cytochromes b5 and P-450, and activities of NADPH-cytochrome c (P-450) reductase (NCR) and 7-ethoxyresorufin O-deethylase (EROD) were measured in liver microsomes prepared from two South American endemic fish, Brycon cephalus and Colossoma macropomum, from tilapia, Oreochromis niloticus, and from Swiss mice, Mus musculus, which served as a control. Strong hemoglobin binding to fish liver microsomal membranes (FLM) altered visible spectra of microsomal cytochromes. Consequently, special precautions during FLM preparation, including liver perfusion followed by repeated washing of microsomes, were required in the study of microsomal cytochromes from these fish. FLM from all fish studied here had a significantly lower content of microsomal cytochromes but a similar level of NCR and EROD activities compared to mouse liver microsomes (MLM). Strong response of the monooxygenase system in O. niloticus to water pollution was detected with both specific cytochrome P-450 content and EROD activity increasing sharply. The optical spectra of hemoglobin from B. cephalus and C. macropomum were analyzed and some differences in shape and relative extinction were observed compared to known hemoglobins.

Membrane effects of trifluoperazine, dibucaine and praziquantel on human erythrocytes.

Trifluoperazine (TFP) is a potent antipsychotic agent, dibucaine (DBC) is a local anaesthetic and praziquantel (PZQ) is a highly effective agent against schistosomiasis. The present work was conducted to (i) investigate the cytotoxic effects of TFP, DBC and PZQ on human erythrocyte membranes; and (ii) compare the alterations induced by the cationic drugs (TFP and DBC) with those induced by the uncharged compound (PZQ), in an attempt to have a better insight on the pathways of each drug-membrane interaction. The erythrocyte morphological alterations induced by sublytic concentrations of TFP, DBC and PZQ were evaluated by scanning electron microscopy and expressed quantitatively by the morphological index. Haemolysis and release of membrane lipids (phospholipids and cholesterol) produced by selected concentrations of TFP, DBC and PZQ, were compared with those resulting from the corresponding triple concentrations of each drug. Our results showed that the uncharged molecule of PZQ induces the same morphological alterations (stomatocytosis) as the cationic drugs TFP and DBC. Haemolysis was shown to vary with the drug used and to be concentration-dependent, with values approximately 10-fold more elevated for TFP and DBC than for PZQ, which revealed a maximum of 6% haemolysis for the highest concentration tested. Different concentration-response curves were obtained for lipid elution, although the profiles of cholesterol and phospholipids released were similar for all drugs. Nevertheless, at a fixed rate of 50% haemolysis, TFP induced a approximately 2-fold increment in the elution of cholesterol when compared with that produced by DBC (P<0. 05). The different effects induced by TFP, DBC and PZQ on erythrocyte morphology, haemolysis and lipid exfoliation are related to the physical and chemical characteristics of each compound. These results suggest that distinct cell membrane interaction pathways lead to drug-specific mechanisms of cytotoxicity.

Pathways involved in trifluoperazine-, dibucaine- and praziquantel-induced hemolysis.

This work elucidates differences in the hemolytic pathway developed by the antipsychotic trifluoperazine (TFP), the local anesthetic dibucaine (DBC) and the antihelminthic praziquantel (PZQ). Their partition coefficients (P) were measured at pH 7.4 between n-octanol, microsomes, liposomes, erythrocyte ghosts and n-octanol/water. The effective drug:lipid molar ratios for the onset of membrane solubilization (ReSAT) and complete hemolysis (ReSOL) were calculated from the experimental P values and compared with a classical surface-active compound treatment Lichtenberg, D. Biochim. Biophys. Acta 821 (1985) 470-478[. The contribution of charged/uncharged forms of TFP and DBC for the hemolytic activity was also analyzed. In all cases the hemolytic phenomena could be related to the monomeric drug insertion into the membrane. Only for TFP at isosmotic condition lysis occurs at concentrations beyond the CMC of the drug, indicating that micellization facilitates TFP hemolytic effect, while DBC and PZQ reach a real membrane saturation at their monomeric form.

Lipid chain dynamics in stratum corneum studied by spin label electron paramagnetic resonance.

The lipid chain motions in stratum corneum (SC) membranes have been studied through electron paramagnetic resonance (EPR) spectroscopy of stearic acid spin-labeled at the 5th, 12th and 16th carbon atom positions of the acyl chain. Lipids have been extracted from SC with a series of chloroform/methanol mixtures, in order to compare the molecular dynamics and the thermotropic behavior in intact SC, lipid-depleted SC (containing covalently bound lipids of the corneocyte envelope) and dispersion of extracted SC lipids. The segmental motion of 5- and 12-doxylstearic acid (5- and 12-DSA) and the rotational correlation time of 16-doxylstearic acid (16-DSA) showed that the envelope lipids are more rigid and the extracted lipids are more fluid than the lipids of the intact SC over the range of temperature measured. The lower fluidity observed for the corneocyte envelope, that may be caused mainly due to lipid-protein interactions, suggests a major contribution of this lipid domain to the barrier function of SC. Changes in the activation energy for reorientational diffusion of the 16-DSA spin label showed apparent phase transitions around 54 degrees C, for the three SC samples. Some lipid reorganization may occur in SC above 54 degrees C, in agreement with results reported from studies with several other techniques. This reorganization is sensitive to the presence of the extractable intercellular lipids, being different in the lipid-depleted sample as compared to native SC and lipid dispersion. The results contribute to the understanding of alkyl chain packing and mobility in the SC membranes, which are involved in the mechanisms that control the permeability of different compounds through skin, suggesting an important involvement of the envelope in the skin barrier.

Contribution of trifluoperazine/lipid ratio and drug ionization to hemolysis.

The interaction of the antipsychotic drug trifluoperazine (TFP) with membranes was investigated in terms of lipid phase perturbation. TFP partition coefficients (P) were measured by phase separation between octanol/water and model membranes/water. The profile of P values at pH 7.4 was: microsomes (7172+/-1229)>liposomes (1916+/-341)>erythrocyte ghosts (1380+/-429)>octanol (452+/-55). Hemolytic experiments showed a biphasic, protective (at lower concentrations) and hemolytic effect above the CMC (42 microM at pH 7.4) of the phenothiazine. By applying classical treatments for surface active compounds to the hemolytic curves, we could calculate P values in whole erythrocyte cells. The preferential binding of uncharged to charged TFP in the membrane was discussed, since it results in a ionization constant (pKapp) different from that observed in the aqueous phase (pK). The TFP ionization constant was decreased from 8.1 (in water) to 7.62 in the presence of membranes and almost the same ratio of charged/uncharged TFP species is present at physiologic pH. Taking into account the DeltapK, we calculated the average TFP partition coefficient between egg phosphatidylcholine liposomes and water, at pH 7.4 (Paverage=1432), which was well correlated with the measured one (Plip=1916). Paverage is highly influenced by the uncharged TFP species and the real base/acid ratio under physiologic conditions was discussed in terms of its possible role in the biological activity of TFP.

Effects of trifluoperazine on the conformation and dynamics of membrane proteins in human erythrocytes.

The chemical modifications induced by trifluoperazine (TFP) in erythrocyte ghosts have been investigated by fluorescence quenching. The apparent distance separating the membrane protein tryptophans and bound 1-aniline-8-naphthalene sulfonate (ANS) molecules decreased after treating erythrocyte membranes with TFP. This effect was accompanied by a significant decrease in the maximum efficiency of energy transfer. We conclude that TFP-induced alterations in the structure of membrane proteins lead to a rearrangement of the surrounding lipids, and consequently to local conformational changes in membrane organization.

Effects of polyoxyethylene chain length on erythrocyte hemolysis induced by poly[oxyethylene (n) nonylphenol] non-ionic surfactants.

The effects of three different poly[oxyethylene (n) nonylphenols], n = 9.5, 20 and 100 oxyethylene (EO) units, on erythrocyte hemolysis and on the fluidity of the erythrocyte membrane were studied. The three different surfactants showed different effects. The surfactant with average n = 9.5 EO units (C9E9) shows a biphasic effect: at low concentrations it protects erythrocytes against hypotonic hemolysis, but at higher concentrations it induces hemolysis both in isotonic and hypotonic buffers. C9E20 does not affect the erythrocyte membrane resistance to hemolysis, independent of the buffer osmolarity; this detergent did not show a hemolytic effect. C9E100 is an effective protective agent against hypotonic hemolysis, in concentration > 2 x 10(-4) M. EPR spectroscopy of spin-labeled stearic acid indicated that the three different surfactants increase the fluidity of erythrocyte ghost membranes. At the higher C9E20 and C9E100 surfactant concentrations in the presence of membrane ghosts, spin-label is located in the surfactant micelles. In the case of the hemolytic concentrations of C9E9, mixed (surfactant plus phospholipid) micelles are formed. These results suggest that C9E9 has a higher affinity for membrane phospholipids, which accounts for its lytic activity. The protective effect of C9E100 is assigned to the osmotic buffering of the liquid surrounding the cell membrane, due to the large polar chains anchored to the membrane outer monolayer but other mechanisms previously considered in the literature may also be effective.

Erythrocruorin of Glossoscolex paulistus (Oligochaeta, Glossoscolecidae): modulation of oxygen affinity by specific antibodies.

1) The Soret region absorption spectrum of erythrocruorin (ERC) obtained from Glossoscolex paulistus, shows that oxy-ERC has a maximum absorption peak at 416 nm while the deoxy-ERC from has a maximum at 427 nm. 2) In the presence of a specific antiserum (anti-ERC) and of anti-ERC immunoglobulin G raised in rabbits, there is a deviation to low wavelengths in the maximum absorption peak of deoxy-ERC while for the oxy form a red-shift is noticed. These shifts accompanied an increased affinity of the hemeprotein for oxygen, possibly because of changes in the overall macromolecular conformation. 3) A decrease in the oxygen affinity of erythrocruorin is observed when large amounts of non-specific serum are used. The same effect is observed in the presence of serum albumin, probably as a result of non-specific binding between the albumin and erythrocruorin. 4) The fluorimetric titration of erythrocruorin with anti-ERC Fab fragments results in a decrease in the intrinsic tryptophan fluorescence of the hemeprotein, a response indicative of a modification in the ERC's quaternary structure.

Water increases the fluidity of intercellular membranes of stratum corneum: correlation with water permeability, elastic, and electrical resistance properties.

We used the spin label electron spin resonance technique to monitor the hydration effect on the molecular dynamics of lipids at C-5, C-12, and C-16 positions of the alkyl chain. Increase in water content of neonatal rat SC leads to an increase in membrane fluidity, especially in the region near the membrane-water interface. The effect is less pronounced deeper inside the hydrophobic core. The reorientational correlation time at the C-16 position of hydrocarbon chains showed a higher change up to approximately 18% (w/w) of water content. This behavior was accompanied by an exponential decay both in elastic modulus and electrical resistance with water content. On the contrary, the segmental motion at C-5 and C-12 positions of the chain and the permeability constant increased in the range of around 18% w/w) up to the fully hydrated condition (58 +/- 7%). Our results give a better characterization of the fluidity of SC and show that it is the principal parameter involved in the mechanism of the permeability of different compounds through skin.

An ESR and spectrophotometric study of the denitration of nitroheterocyclic drugs by liver homogenates and their metabolic consumption by liver microsomes from cytochrome P-450-induced mice.

This work extends a previous study on the mechanism of hepatic denitration of two nitroheterocyclic drugs (NHCD), quinifuryl and nitracrine, in which the release of nitric oxide (NO) from these compounds can be accompanied by the formation of a NO-heme iron complex. Pretreating mice with three inducers of cytochrome P-450 (phenobarbital, clophen A50 and butylated hydroxytoluene (BHT)) increased the yield of the nitrosyl complex which correlated with a rise in the cytochrome P-450 content of mouse liver microsomes. In contrast, treating the animals with beta-naphthoflavone decreased the complex yield while still increasing P-450 content. Treating the animals with any of the above inducers significantly increased the rate of NHCD metabolism in mouse liver microsomes. Based on these results, a possible mechanism for hepatic NHCD denitration is discussed.

Effect of hydration upon the fluidity of intercellular membranes of stratum corneum: an EPR study.

The principal mechanisms controlling the molecular permeability through the skin are associated to the intercellular membranes of stratum corneum (SC), the outermost layer of mammalian skin. It is generally accepted that an increase in fluidity of these membranes leads to a reduction of the physical barrier exerted by SC with a consequent enhancement in permeation of different compounds. It is known that water diffusion in SC increases with the increase in the water content in SC. Using the spin labeling method we evaluate the effect of hydration on the fluidity of intercellular membranes at three depths of the alkyl chain. Increase in the water content in SC leads to a drastic increase in membrane fluidity especially in the region near the membrane/water interface; the effect decreases on going deeper inside the hydrophobic core. Analysis of electron paramagnetic resonance (EPR) parameters as a function of temperature showed that the rotational motion at depth of the 16th carbon atom of the chain experienced a phase transition at 45 and 60 degrees C. These phase transition temperatures were not altered by changes in the water content of SC. A phase transition between 28 and 48 degrees C was observed from the segmental motion in the region near the polar headgroup (up to 12th carbon in the chain) and was strongly dependent upon the hydration of SC. Our results give a better characterization of the fluidity of SC, the main parameter involved in the mechanisms that control the permeability of different compounds through skin.

Stratum corneum intercellular lipid as compared to erythrocyte ghosts: an ESR study of thermotropic behavior and nitroxide reduction. Electron Spin Resonance.

Intercellular membranes of Stratum Corneum and erythrocyte ghosts were studied through partition of Tempo nitroxide spin label. Stratum Corneum presents a phase transition-monitored by the spin label at 58 degrees C--that is in agreement with data obtained through the use of other techniques. A reduction of the nitroxide is observed. Erythrocyte ghosts at protein concentrations higher than the values generally used in ESR studies (above 15 mg/ml) also show the partition and reduction of Tempo. Experimental data suggest that the reduction mechanism might be common to both types of membranes involving probably the membrane lipoperoxidation as well as protein oxidation.

Mouse liver microsomes (MLM) protect erythrocytes against trifluoperazine (TFP) induced and mechanical hemolysis which are due to TFP microsomal transformation and to the action of an unidentified water-soluble microsomal factor (UF).

Trifluoperazine (TFP), a phenothiazine derivative, produces either hemolysis or protection of erythrocytes under isosmotic conditions in a dose-dependent manner. The hemolytic effect of TFP is abolished in the presence of mouse liver microsomes (MLM) which is due, in part, to drug incorporation, transformation and a MLM enzyme driven metabolism. An unidentified water-soluble factor (or factors) derived from MLM has been found to protect erythrocytes against both mechanical and TFP-induced isosmotic hemolysis.

Interactions between vasodilator drugs and human hemoglobin.

1. The in vitro effect of the vasodilators papaverine, dipyridamole, nifedipine, prenylamine and glyceryl di-, tri- and tetranitrate on the oxygen saturation curve of human hemoglobin (Hb) was evaluated. The Hb dissociation curve was measured spectrophotometrically both for freshly obtained heparinized blood and for purified Hb A after incubation with the drugs. 2. For both purified Hb and whole blood samples, incubation with papaverine, dipyridamole and nifedipine modified P50 (PO2 for half saturation of the Hb/oxygen sites) at pH 7.4: 30.9 mmHg for 0.72 mM papaverine vs 23.9 mmHg for control; 23.9 mmHg for 1.43 mM dipyridamole vs 20.8 mmHg for control; 1.09 mmHg for 2.73 mM nifedipine vs 1.14 mmHg for control stripped Hb. The effects of the three vasodilators on Hb were correlated with pH, methemoglobin formation, temperature and incubation time. 3. P50 values were determined for Hb of the blood of angina patients treated with one of the vasodilators, and blood 2,3-diphosphoglycerate (2,3-DPG) levels were also measured. In contrast to the in vitro data, neither P50 nor 2,3-DPG levels were significantly modified in the blood of patients treated with one of the vasodilators. 4. These in vitro studies demonstrated that the vasodilators papaverine and dipyridamole decrease, and nifedipine increases the oxygen affinity of Hb in red blood cells or as a purified hemoglobin.

Interactions between vasodilator drugs and human hemoglobin

The in vitro effect of the vasodilators papaverine, dipyridamole, nifedipine, prenylamine and glyceryl di-tri- and tetranitranitrate on the oxygen saturation curve of human hemoglobin (Hb) was evaluated. The Hb dissociation curve was measured spectrophotometrically both for freshly obtained heparinized blood and for purified Hb A after incubation with the drugs. For both purified Hb and whole blood samples, incubation with papaverine, dipyridamole and nifedipine modified P50 (PO2 for half saturation of the Hb/oxygen sites) at pH 7.4: 30.9 mmHg for 0.72 mM papaverine vs 23.9 mmHg for control; 23.9 mmHg for 1.43 mM dipyridamole vs 20.8 mmHg for control; 1.09 mmHg for 2.73 mM nifedipine vs 1.14 mmHg for control stripped Hb. The effects of the three vasodilators on Hb were correlated with pH, methemoglobin formation, temperature and incubation time. P50 values were determined for Hb of the blood of angina patients treated with one of the vasodilators, and blood 2,3-diphosphoglycerate (2,3-DPG) levels were also measured. In contrast to the in vitro data, neither P50 nor 2,3-DPG levels were significantly modified in the blood of patients treated with one of the vasodilators. These in vitro studies demonstrated that the vasodilators papaverine and dipyridamole decrease, and nifedipine increases the oxygen affinity of Hb in red blood cells or as a purified hemoglobin

Anomalous pressure dissociation of large protein aggregates. Lack of concentration dependence and irreversibility at extreme degrees of dissociation of extracellular hemoglobin.

The effects of hydrostatic pressure on the extracellular hemoglobin of Glossoscolex paulistus were investigated by studies of light scattering, intrinsic protein fluorescence, filtration chromatography, and oxygen binding. Pressure promoted a large decrease of light scattering consistent with the dissociation of the hemoglobin. Pressures up to 1.7 kbar caused dissociation with reversibility of the light scattering and fluorescence properties after return to atmospheric pressure. Higher pressures provoked additional dissociation with increasing loss of reversibility. After complete dissociation by incubation at 2.5 kbar followed by decompression, the protein continued mostly dissociated. The dissociated forms were distributed in two populations as based on size exclusion chromatography, one corresponding to small dissociated units (average Mr = 33,000) and the other population corresponding to the one-twelfth subunit (260,000 Mr). The pressure dissociation curves showed no significant dependence on protein concentration suggesting that the native hemoglobin population exists in a distribution of free-energies of association. Both the decrease of concentration dependence and the loss of ability to reassemble seem to increase with the complexity and size of the protein aggregate. These findings permit the conclusion that increased heterogeneity of free-energies of association with the size of the aggregate may result in the molecular individuality of large protein complexes such as subcellular particles and viruses.