A role for transforming growth factor-beta apoptotic signaling pathway in liver injury induced by ingestion of water contaminated with high levels of Cr(VI).

Hexavalent chromium [Cr(VI)] exposure is commonly associated with lung cancer. Although other adverse health effects have been reported, some authors, on assuming that orally ingested Cr(VI) is efficiently detoxified upon reduction by body fluids, believe that Cr(VI) do not target cells other than respiratory tract cells. In rodents, ingested Cr(VI)-contaminated water was reported to induce, in the liver, increases in TGF-beta transcripts. As TGF-beta dependent signaling pathways are closely associated with hepatic injury, the present study was undertaken addressing two specific issues: the effects of ingestion of water contaminated with high levels of Cr(VI) in rat liver structure and function; and the role of the TGF-beta pathway in Cr(VI)-induced liver injury. Examination of Wistar rats exposed to 20 ppm Cr(VI)-contaminated water for 10 weeks showed increased serum glucose and alanine aminotransferase (ALT) levels. Liver histological examination revealed hepatocellular apoptosis, further confirmed by immunohystochemical study of Caspase 3 expression. Liver gene expression analysis revealed increased expression of Smad2/Smad4 and Dapk, suggesting the involvement of the TGF-beta pathway in the apoptotic process. Since no changes in Smad3 expression were observed it appears apoptosis is using a Smad3-independent pathway. Increased expression of both Caspase 8 and Daxx genes suggests also the involvement of the Fas pathway. Gene expression analysis also revealed that a p160(ROCK)-Rho-independent pathway operates, leading to cell contraction and membrane blebbing, characteristic apoptotic features. These findings suggest that either the amount of Cr(VI) ingested overwhelmed the body fluids reductive capacity or some Cr(VI) escapes the reductive protection barrier, thus targeting the liver and inducing apoptosis.

Comparative study of tributyltin toxicity on two bacteria of the genus Bacillus.

Tributyltin is a potent biocide mainly used in marine anti-fouling paints. Owing to its widespread distribution in coast areas and its high toxicity to aquatic organisms, the use of this compound is generally restricted and under government regulation. Despite of that, it persists in the aquatic environment. Organotins used in industry have also been detected in terrestrial environments. The persistence and high lipophilicity explain bioaccumulation. The role of bacteria in recycling organic matter prompted us to study the interaction of tributyltin with two ubiquitous bacilli, B. stearothermophilus and B. subtilis, proposed as biological indicators of pollutants with ecological impact. These bacteria have been used as suitable models for the study of toxicity mechanisms of unselective lipophilic compounds (e.g., DDT and endosulfan). Drug effects on growth parameters, oxygen consumption and membrane organization were assessed. Bacteria growth in a liquid complex medium was disturbed by concentrations of TBT as low as 25 nM (8 microgL(-1)), close to the concentration in polluted environments. The respiratory activity is affected by TBT in both microorganisms. Membrane organization, assessed by fluorescence polarization of two fluidity probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and a propionic acid derivative (DPH-PA), was also perturbed by the xenobiotic. Alterations on growth, oxygen consumption and physical properties of membrane lipids are stronger in B. stearothermophilus as compared to B. subtilis. A putative relationship between growth inhibition and respiratory activity impairment induced by TBT and its effects on the physical behaviour of bacterial membrane lipids is suggested.

Toxicity of methoprene as assessed by the use of a model microorganism.

Methoprene is an insect juvenile growth hormone mimic, commonly used as a pesticide. Although widely used for the control of several pests, toxic effects on organisms of different phyla have been reported. These events triggered studies to clarify the mechanisms of toxicity of this insecticide putatively involved in ecological issues. Here we show the effect of methoprene on the normal cell growth and viability of a strain of the thermophilic eubacterium Bacillus stearothermophilus, previously used as a model for toxicological evaluation of other environment pollutants. Respiration studies were also carried out attempting to identify a putative target for the cytotoxic action of methoprene. Cell growth was affected and a decrease of the number of viable cells was observed as a result of the addition of methoprene to the growth medium, an effect reverted by the presence of Ca(2+). Methoprene also inhibited the redox flow of B. stearothermophilus protoplasts before the cytochrome oxidase segment, an effect further studied by individually assessing the enzymatic activities of the respiratory complexes. This study suggests that methoprene membrane interaction and perturbation of cell bioenergetics may underlie the mechanism of toxicity of this compound in non-target organisms.

Use of the microorganism Bacillus stearothermophilus as a model to evaluate toxicity of the lipophilic environmental pollutant endosulfan.

Microorganisms are very powerful tools for the supply of information about the toxic effects of lipophilic compounds, since an impairment of cell growth usually occurs as a result of perturbations related, in most cases, with the partition of toxicants in membranes. The thermophilic eubacterium Bacillus stearothermophilus has been used as a model system to identify alpha- and beta-endosulfan interactions with the membrane possibly related with the insecticide toxicity. Two approaches have been pursued: (a) bacterial growth is followed and the effects of endosulfan isomers determined; (b) biophysical studies with the fluorescent fluidity probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to assess the effects of alpha- and beta-endosulfan on the organization of the membrane lipid bilayer. The effects on growth were quantitatively evaluated by determination of growth parameters, namely the lag phase, the specific growth rate and the cell density reached by cultures in the stationary phase. Growth inhibition by alpha and beta-endosulfan dependent on the concentration is diminished or removed by the addition of 2.5 mM Ca2+ to bacterial cultures. Fluorescence DPH polarization consistently showed opposite effects of Ca2+ and alpha- and beta-endosulfan on the physical state of bacterial polar lipid dispersions.

Molecular mechanisms of the metabolite 4-hydroxytamoxifen of the anticancer drug tamoxifen: use of a model microorganism.

A strain of the thermophilic eubacterium Bacillus stearothermophilus was used as a model system to identify membrane mediated cytotoxic effects of 4-hydroxytamoxifen, following previous studies with tamoxifen. With this experimental approach we attempted to further clarify tamoxifen and 4-hydroxytamoxifen membrane interactions often evoked as responsible for their multiple cellular effects. Bacterial growth and the oxygen consumption rate provided quantitative data of the cytotoxic action of hydroxytamoxifen. The effects of hydroxytamoxifen on the physical properties of bacterial lipid membrane preparations were also evaluated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Cultures of B. stearothermophilus grown in a complex medium containing hydroxytamoxifen in the concentration range of 1 to 7 microM exhibited progressively longer lag adapting periods, decreased specific growth rates and lower growth yields, as compared to control cultures. Hydroxytamoxifen also affected the electron redox flow of B. stearothermophilus protoplasts and induced significant perturbation of the structural order of bacterial lipid dispersions. We concluded that the bacterial model provides useful information about the nature and repercussion of membrane physical interactions of this lipophilic drug, on the basis of an easy and economic methodology.

Chromium(VI) interaction with plant and animal mitochondrial bioenergetics: a comparative study.

The mechanism of Cr(VI)-induced toxicity in plants and animals has been assessed for mitochondrial bioenergetics and membrane damage in turnip root and rat liver mitochondria. By using succinate as the respiratory substrate, ADP/O and respiratory control ratio (RCR) were depressed as a function of Cr(VI) concentration. State 3 and uncoupled respiration were also depressed by Cr(VI). Rat mitochondria revealed a higher sensitivity to Cr(VI), as compared to turnip mitochondria. Rat mitochondrial state 4 respiration rate triplicated in contrast to negligible stimulation of turnip state 4 respiration. Chromium(VI) inhibited the activity of the NADH-ubiquinone oxidoreductase (complex I) from rat liver mitochondria and succinate-dehydrogenases (complex II) from plant and animal mitochondria. In rat liver mitochondria, complex I was more sensitive to Cr(VI) than complex II. The activity of cytochrome c oxidase (complex IV) was not sensitive to Cr(VI). Unique for plant mitochondria, exogenous NADH uncoupled respiration was unaffected by Cr(VI), indicating that the NADH dehydrogenase of the outer leaflet of the plant inner membrane, in addition to complexes III and IV, were insensitive to Cr(VI). The ATPase activity (complex V) was stimulated in rat liver mitochondria, but inhibited in turnip root mitochondria. In both, turnip and rat mitochondria, Cr(VI) depressed mitochondrial succinate-dependent transmembrane potential (Deltapsi) and phosphorylation efficiency, but it neither affected mitochondrial membrane permeabilization to protons (H+) nor induced membrane lipid peroxidation. However, Cr(VI) induced mitochondrial membrane permeabilization to K+, an effect that was more pronounced in turnip root than in rat liver mitochondria. In conclusion, Cr(VI)-induced perturbations of mitochondrial bioenergetics compromises energy-dependent biochemical processes and, therefore, may contribute to the basal mechanism underlying its toxic effects in plant and animal cells.

Hydroxytamoxifen interaction with human erythrocyte membrane and induction of permeabilization and subsequent hemolysis.

4-Hydroxytamoxifen (OHTAM) is the most active metabolite of the widely prescribed anticancer drug tamoxifen (TAM) used in breast cancer therapy. This work describes the effects of OHTAM on isolated human erythrocytes, using standardized test conditions, to check for a putative contribution to the TAM-induced hemolysis and to study basic mechanisms involved in the interaction of OHTAM with cell membranes. Incubation of isolated human erythrocytes with relatively high concentrations of OHTAM results in a concentration-dependent hemolysis, its hemolytic effect being about one-third of that induced by TAM. OHTAM-induced hemolysis is prevented by either alpha-tocopherol (alpha-T) or alpha-tocopherol acetate (alpha-TAc) and it occurs in the absence of oxygen consumption and hemoglobin oxidation, ruling out the oxidative damage of erythrocytes. However, OHTAM remarkably increases the osmotic fragility of erythrocytes, increasing the susceptibility of erythrocytes to hypotonic lysis. Additionally, the hemoglobin release induced by OHTAM is preceded by a rapid efflux of intracellular K(+). Therefore, our data suggest that OHTAM-induced hemolysis does not contribute to TAM-induced hemolytic anemia and it is a much weaker toxic drug as compared with TAM. Moreover, at variance with the membrane disrupting effects of TAM, OHTAM promotes perturbation of the membrane's backbone region due to its strong binding to proteins with consequent formation of membrane paths of permeability to small solutes and retention of large solutes like hemoglobin, followed by osmotic swelling and cell lysis. The prevention of OHTAM-induced hemolysis by alpha-T and alpha-TAc is probably committed to the permeability sealing resulting from structural stabilization of membrane.

Mitochondrial permeability transition induced by the anticancer drug etoposide.

Etoposide (VP-16) is widely used for the treatment of several forms of cancer. The cytotoxicity of VP-16 has been assigned to the induction of apoptotic cell death but the signaling pathway for VP-16-induced apoptosis is essentially unknown. There is some evidence that this process depends on events associated with the loss of mitochondrial membrane potential (Delta Psi) and/or release of apoptogenic factors, putatively as a consequence of mitochondrial permeability transition (MPT) induction. This work evaluates the interference of VP-16 with MPT in vitro, which is characterized by the Ca(2+)-dependent depolarization of Delta Psi, the release of matrix Ca(2+) and by extensive swelling of mitochondria. Delta Psi depolarization and Ca(2+) release were measured with ion-selective electrodes, and mitochondrial swelling was monitored spectrophotometrically. Incubation of rat liver mitochondria with VP-16 results in a concentration-dependent induction of MPT, evidenced by an increased sensitivity to Ca(2+)-induced swelling, depolarization of Delta Psi, Ca(2+) release by mitochondria and stimulation of state 4 oxygen consumption. All of these effects are prevented by preincubating the mitochondria with cyclosporine A, a potent and specific inhibitor of the MPT. Therefore, VP-16 increases the sensitivity of isolated mitochondria to the Ca(2+)-dependent induction of the MPT. Together, these data provide a possible mechanistic explanation for the previously reported effects of VP-16 on apoptosis induction.

Toxic effects of tamoxifen on the growth and respiratory activity of Bacillus stearothermophilus.

The anticancer drug tamoxifen (TAM) is used as first line therapy in breast cancer. Although tamoxifen is usually considered an estrogen antagonist, several studies suggest alternative mechanisms of action. Bacillus stearothermophilus has been used as a model to clarify the antiproliferative action of tamoxifen putatively related with drug-membrane interaction. According to previous data, TAM induces perturbation of membrane structure along with impairment of bacterial growth. The aim of this work was to correlate the effects of TAM on growth of intact B. stearothermophilus with the respiratory activity of isolated protoplasts of this bacteria. TAM inhibits bacterial growth and oxygen consumption of protoplasts as a function of concentration. Effects on oxygen consumption depend on the substrate used: NADH, allowing to study the full respiratory chain and ascorbate-TMPD to probe the final oxidase segment. The interaction of TAM with the respiratory components occurs at a level preceding the cytochrome oxidase segment.

Ethylazinphos interaction with membrane lipid organization induces increase of proton permeability and impairment of mitochondrial bioenergetic functions.

Ethylazinphos increases the passive proton permeability of lipid bilayers reconstituted with dipalmitoylphosphatidylcholine (DPPC) and mitochondrial lipids. A sharp increase of proton permeability is detected at insecticide/lipid molar ratios identical to those inducing phase separation in the plane of DPPC bilayers, as revealed by differential scanning calorimetry (DSC). Ethylazinphos progressively depresses the transmembrane potential (DeltaPsi) of mitochondria supported by piruvate/malate, succinate, or ascorbate/TMPD. Additionally, a decreased depolarization induced by ADP depends on ethylazinphos concentration, reflecting a phosphorylation depression. This loss of phosphorylation is a consequence of a decreased DeltaPsi. A decreased respiratory control ratio is also observed, since ethylazinphos stimulates state 4 respiration and inhibits ADP-stimulated respiration (state 3). Ethylazinphos concentrations up to 100 nmol/mg mitochondrial protein increase the rate of state 4 together with a decrease in DeltaPsi, without significant perturbation of state 3 and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled respiration. For increased insecticide concentrations, the state 3 and FCCP-uncoupled respiration are inhibited to approximately the same extent. The perturbations are more pronounced when the energization is supported by pyruvate/malate and less effective when succinate is used as substrate. The present data, in association with previous DSC studies, indicate that ethylazinphos, at concentrations up to 100 nmol/mg mitochondrial protein, interacts with the lipid bilayer of mitochondrial membrane, changing the lipid organization and increasing the proton permeability of the inner membrane. The increased proton permeability explains the decreased oxidative phosphorylation coupling. Resulting disturbed ATP synthesis may significantly underlie the mechanisms of ethylazinphos toxicity, since most of cell energy in eukaryotes is provided by mitochondria.

Changes induced by malathion, methylparathion and parathion on membrane lipid physicochemical properties correlate with their toxicity.

Perturbations induced by malathion, methylparathion and parathion on the physicochemical properties of dipalmitoylphosphatidylcholine (DPPC) were studied by fluorescence anisotropy of DPH and DPH-PA and by differential scanning calorimetry (DSC). Methylparathion and parathion (50 microM) increased the fluorescence anisotropy evaluated by DPH-PA and DPH, either in gel or in the fluid phase of DPPC bilayers, but mainly in the fluid phase. Parathion is more effective than methylparathion. On the other hand, malathion had almost no effect. All the three xenobiotics displaced the phase transition midpoint to lower temperature values and broadened the phase transition profile of DPPC, the effectiveness following the sequence: parathion>methylparathion>>malathion. A shifting and broadening of the phase transition was also observed by DSC. Furthermore, at methylparathion/lipid molar ratio of 1/2 and at parathion/lipid molar ratio of 1/7, the DSC thermograms displayed a shoulder in the main peak, in the low temperature side, suggesting coexistence of phases. For higher ratios, the phase transition profile becomes sharp as the control transition, but the midpoint is shifted to the previous shoulder position. Conversely to methylparathion and parathion, malathion did not promote phase separation. The overall data from fluorescence anisotropy and calorimetry indicate that the degree of effect of the insecticides on the physicochemical membrane properties correlates with toxicity to mammals. Therefore, the in vivo effects of organophosphorus compounds may be in part related with their ability to perturb the phospholipid bilayer structure, whose integrity is essential for normal cell function.

Differential sensitivities of plant and animal mitochondria to the herbicide paraquat.

Paraquat herbicide is toxic to animals, including humans, via putative toxicity mechanisms associated to microsomal and mitochondrial redox systems. It is also believed to act in plants by generating highly reactive oxygen free radicals from electrons of photosystem I on exposure to light. Paraquat also acts on non-chlorophyllous plant tissues, where mitochondria are candidate targets, as in animal tissues. Therefore, we compared the interaction of paraquat with the mitochondrial bioenergetics of potato tuber, using rat liver mitochondria as a reference. Paraquat depressed succinate-dependent mitochondrial Delta(psi), with simultaneous stimulation of state 4 O2 consumption. It also induced a slow time-dependent effect for respiration of succinate, exogenous NADH, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)/ascorbate, which was more pronounced in rat than in potato mitochondria. However, with potato tuber mitochondria, the Delta(psi) promoted by complex-I-dependent respiration is insensitive to this effect, indicating a protection against paraquat radical afforded by complex I redox activity, which was just the reverse of to the findings for rat liver mitochondria. The experimental set up with the tetraphenyl phosphonium (TPP+)-electrode also indicated production of the paraquat radical in mitochondria, also suggesting its accessibility to the outside space. The different activities of protective antioxidant agents can contribute to explain the different sensitivities of both kinds of mitochondria. Values of SOD activity and alpha-tocopherol detected in potato mitochondria were significantly higher than in rat mitochondria, which, in turn, revealed higher values of lipid peroxidation induced by paraquat.

Lipid composition and dynamics of cell membranes of Bacillus stearothermophilus adapted to amiodarone.

Bacillus stearothermophilus, a useful model to evaluate membrane interactions of lipophilic drugs, adapts to the presence of amiodarone in the growth medium. Drug concentrations in the range of 1-2 microM depress growth and 3 microM completely suppresses growth. Adaptation to the presence of amiodarone is reflected in lipid composition changes either in the phospholipid classes or in the acyl chain moieties. Significant changes are observed at 2 microM and expressed by a decrease of phosphatidylethanolamine (relative decrease of 23.3%) and phosphatidylglycerol (17.9%) and by the increase of phosphoglycolipid (162%). The changes in phospholipid acyl chains are expressed by a decrease of straight-chain saturated fatty acids (relative decrease of 12.2%) and anteiso-acids (22%) with a parallel increase of the iso-acids (9.8%). Consequently, the ratio straight-chain/branched iso-chain fatty acids decreases from 0. 38 (control cultures) to 0.30 (cultures adapted to 2 microM amiodarone). The physical consequences of the lipid composition changes induced by the drug were studied by fluorescence polarization of diphenylhexatriene and diphenylhexatriene-propionic acid, and by differential scanning calorimetry. The thermotropic profiles of polar lipid dispersions of amiodarone-adapted cells are more similar to control cultures (without amiodarone) than those resulting from a direct interaction of the drug with lipids, i.e., when amiodarone was added directly to liposome suspensions. It is suggested that lipid composition changes promoted by amiodarone occur as adaptations to drug tolerance, providing the membrane with physico-chemical properties compatible with membrane function, counteracting the effects of the drug.

Toxicity assessment of tamoxifen by means of a bacterial model.

A strain of Bacillus stearothermophilus was used as a model to study physical perturbations induced in the membrane by the cytostatic tamoxifen (TAM). This study was carried out using two lines of criteria: (1) bacterial growth, and temperature growth range, with determination of growth parameters as a function of TAM concentration; and (2) biophysical studies by differential scanning calorimetry (DSC) and by means of two fluorescent probes to evaluate perturbations promoted by the drug on the structural order of bacterial lipid membranes. The inhibition of growth induced by TAM, the structural bilayer disordering, and the shift in the phase transition temperature to a lower range were also determined in the presence of Ca2+, i.e., a natural membrane stabilizer, to elucidate further perturbing effects of TAM on membranes with putative implications in cell toxicity. Growth inhibition promoted by TAM is potentiated by an increase in growth temperature above the optimal range, but attenuated or relieved by the addition of 2.5 mM Ca2+ to the culture medium. Consistently, fluorescence polarization and DSC studies showed that Ca2+ ions (2.5 mM) effectively compensated for the destabilizing effects promoted by TAM in bacterial lipid membranes.

Membrane lipid composition of Bacillus stearothermophilus as affected by lipophilic environmental pollutants: an approach to membrane toxicity assessment.

The thermophilic eubacterium Bacillus stearothermophilus is used as a model to identify membrane perturbing effects of lipophilic compounds. A parallelism has been established between the toxicity of the organochlorine insecticide DDT and its metabolite, DDE, in bacterial growth and the effects on cell functions and physical perturbations induced at the membrane (Donato et al. 1997a, Arch Environ Contam Toxicol 33:109-116; Donato et al. 1997b, Appl Environ Microbiol 63:4948-495). In the present work, the use of B. stearothermophilus as a model of screening for chemical toxicity has been implemented. Because the regulation of the lipid composition of the membrane is a common strategy in response to adverse growth conditions, we studied the effects of DDE on the lipid composition and the consequent alterations of membrane physical properties in comparison to the parental compound DDT. As expected, different adaptation responses were induced by the compounds, being DDT more effective as compared with DDE. Collected data are consistent with the stronger perturbations induced by DDT on growth and membrane functions. It is concluded that the membrane lipid composition of the bacterium is a very sensitive criterium to detect membrane-mediated toxic effects at low concentrations of lipophilic xenobiotics.

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure.

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.

Occurrence of plant-uncoupling mitochondrial protein (PUMP) in diverse organs and tissues of several plants.

The presence of plant-uncoupling mitochondrial protein (PUMP), previously described by Vercesi et al. (1995), was screened in mitochondria of various organs or tissues of several plant species. This was done functionally, by monitoring purine nucleotide-sensitive linoleic acid-induced uncoupling, or by Western blots. The following findings were established: (1) PUMP was found in most of the higher plants tested; (2) since ATP inhibition of linoleic acid-induced membrane potential decrease varied, PUMP content might differ in different plant tissues, as observed with mitochondria from maize roots, maize seeds, spinach leaves, wheat shoots, carrot roots, cauliflower, broccoli, maize shoots, turnip root, and potato calli. Western blots also indicated PUMP presence in oat shoots, carnation petals, onion bulbs, red beet root, green cabbage, and Sedum leaves. (3) PUMP was not detected in mushrooms. We conclude that PUMP is likely present in the mitochondria of organs and tissues of all higher plants.

Perturbations induced by alpha- and beta-endosulfan in lipid membranes: a DSC and fluorescence polarization study.

The interaction of alpha- and beta-endosulfan isomers with lipid bilayers was searched by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6- and 12-(9-anthroyloxy) stearic acids (2-AS, 6-AS and 12-AS) and 16-(9-anthroyloxy) palmitic acid (16-AP). Both endosulfan isomers, at insecticide/lipid molar ratios ranging from 1/40 to 1/1, shift the phase transition midpoint to lower temperature values and broaden the transition profile of dipalmitoylphosphatidylcholine (DPPC) bilayers. At insecticide/lipid molar ratios of 1/40, the isomers fully abolish the bilayer pretransition. Conversely to beta-endosulfan, alpha-endosulfan promotes a new phase transition, centered at 35.4 degrees C, in addition to the main phase transition of DPPC. Therefore, the alpha-isomer may undergo a heterogeneous distribution in separate domains in the plane of the membrane, whereas the beta-isomer may undergo a homogeneous distribution. Fluorescence polarization data indicate that alpha-endosulfan increases the lipid structural order in the regions probed by 2-AS and decreases it in the regions probed by 6-AS, 12-AS and 16-AP. On the other hand, the beta-isomer produces disordering effects in the upper regions of the bilayers, probed by 2-AS, and ordering in deeper regions, probed by 6-AS, 12-AS and 16-AP, mainly in the gel phase. The incorporation of cholesterol into DPPC bilayers progressively decreases the effects of beta-isomer which are vanished at 20 mol% cholesterol. However, this and higher cholesterol concentrations did not prevent alpha-endosulfan membrane interaction, as revealed by DSC and fluorescence polarization. The distinct effects promoted by alpha- and beta-endosulfan are discussed in terms of molecular orientation and positioning within the bilayer. Apparently, the alpha-isomer preferentially locates closer to the phospholipid headgroups whereas the beta-isomer distributes in deeper domains of the bilayer.

Neutrality of amiodarone on the initiation and propagation of membrane lipid peroxidation.

Amiodarone is an iodinated benzofuran derivative largely used as an antiarrhythmic. Owing to the sensitivity of heart tissue to radicals, amiodarone was assayed for putative effects on lipid peroxidation studied in liposomes of soybean phosphatidylcholine and of bovine heart mitochondrial lipids used as model systems. Lipid peroxidations were initiated with Fe2+/ascorbic acid, and with peroxyl radicals generated from the azocompounds, AAPH and AMVN. These assays were carried out by following the quenching of the fluorescent probe cis-parinaric acid and by monitoring oxygen consumption. It has been ascertained that amiodarone does not protect or potentiate significantly the lipid peroxidation both lipidic systems. To fully ascertain the neutral behaviour of amiodarone in the lipid peroxidation process, the degradation of phospholipid acyl chains has been checked by GLC. These data confirm that amiodarone does not protect or potentiate lipid peroxidation to a significant extent. It is concluded that the limited effects of amiodarone might be related only indirectly with the lipid peroxidation. It is possible that the drug causes limited conformational and biophysical alterations in membrane phospholipid bilayers that can affect the process of peroxidation. Therefore, it is concluded that the therapeutic effects and benefits as a heart antiarrhythmic agent are independent of lipid peroxidation processes. Furthermore, the interaction of the drug with lipid bilayers does not induce significant conformational perturbations that could significantly favour or depress the peroxidation process.

Biophysical perturbations induced by ethylazinphos in lipid membranes.

Perturbations induced by ethylazinphos on the physical organization of dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol membranes were studied by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6-, 12-(9-anthroyloxy) stearic acids and 16-(9-anthroyloxy) palmitic acid. Ethylazinphos (50 and 100 microM) increases the fluorescence polarization of the probes, either in the gel or in the fluid phase of DPPC bilayers, and this concentration dependent effect decreases from the surface to the bilayer core. Additionally, the insecticide displaces the phase transition to a lower temperature range and broadens the transition profile of DPPC. A shifting and broadening of the phase transition is also observed by DSC. Furthermore at insecticide/lipid molar ratios higher than 1/7, DSC thermograms, in addition to the normal transition centered at 41 degrees C, also display a new phase transition centered at 45.5 degrees C. The enthalpy of this new transition increases with insecticide concentration, with a corresponding decrease of the main transition enthalpy. Ethylazinphos in DPPC bilayers with low cholesterol (< or = 20 mol%) perturbs the membrane organization as described above for pure DPPC. However, cholesterol concentrations higher than 20 mol% prevent insecticide interaction, as revealed by fluorescence polarization and DSC data. Apparently, cholesterol significantly modulates insecticide interaction by competition for similar distribution domains in the membrane. The present results strongly support our previous hypothesis that ethylazinphos locates in the cooperativity region, i.e. the region of C1-C9 atoms of the acyl chains, and extends to the lipid-water interface, where it increases lipid packing order sensed across all the thickness of the bilayer. Additionally, and, on the basis of DSC data, a lateral regionalization of ethylazinphos is here tentatively suggested.