Disaccharide modulation of the mitochondrial membrane fluidity changes induced by the membrane potential.

The influence of the medium composition on the dynamic properties of mitochondrial membranes on depolarization was studied by following the fluorescence anisotropy changes of mitochondria-bound 1,6-diphenyl-1,3,5-hexatriene (DPH) and hematoporphyrin (HP) as reporters, respectively, of lipid and protein regions. On collapse of the potential, the membrane fluidity increased in NaCl-, KCl-, and monosaccharide-based media and decreased in disaccharides. Infrared spectroscopy experiments suggested that disaccharides likely change water's structure and association on the membrane surface. These results indicate that disaccharides induce membrane perturbation, which may interfere in the study of structure-function correlation in biological membranes.

The effects of singlet oxygen produced by photodynamic action on the mitochondrial permeability transition differ in accordance with the localization of the sensitizer.

We have examined whether the effects of singlet oxygen (1O2) produced by photodynamic action on the mitochondrial permeability transition (PT) can be modulated by the localization of photosensitizers in irradiated mitochondria. We have previously shown that oxidation due to 1O2 photogenerated in hematoporphyrin (HP)-loaded mitochondria can prevent opening of the PT pores, likely after degradation of some critical histidines (Salet et al, 1997, J. Biol. Chem. 272, 21938-21943). Equally, in the present study we have irradiated mitochondria in the presence of a structurally different photosensitizer producing 1O2, namely 4,5',8-trimethylpsoralen (TMP). Fluorescence studies show that TMP binds to protein sites which differ from those of HP. In sharp contrast with HP, TMP-driven photodynamic action triggers per se pore opening. Interestingly, this inducing effect is inhibited when TMP-treated mitochondria are irradiated after addition of mersalyl, a specific reagent protecting thiol groups of the inner mitochondrial membrane that are oriented toward the external hydrophilic phase. This fact suggests that 1O2-mediated thiol oxidation is responsible for TMP-photoinduced pore opening. Taken together, these findings suggest that 1O2 can activate or inactivate a cellular function such as mitochondrial PT depending on the site where it is produced in the mitochondrial membrane.

Effects of the lipophilic biscation, bis-pyridinium oxime BP12, on bioenergetics and induction of permeability transition in isolated mitochondria.

In order to further investigate the mechanism of action of bridged lipophilic bis-pyridinium oximes previously observed to interfere with mitochondrial metabolism and to induce growth arrest and apoptosis in HeLa cells (Nocentini et al., Biochem Pharmacol 53: 1543-1552, 1997), we studied the effects of a bis-pyridinium oxime with a polymethylene chain N = 12 (BP12) on isolated rat liver mitochondria. Respiration in the absence of ADP with succinate plus rotenone as substrate was not affected after treatment with various concentrations of BP12 up to 10 microM, while the ADP-stimulated respiration was slowed down, with a parallel decrease in ATP synthesis. No effects of BP12 were detected on membrane potential, ATPase activity, and inorganic phosphate transport, but the adenine nucleotide translocase was inactivated and a permeability transition of the inner membrane was induced in the presence of calcium. These data suggest that mitochondrial impairment of ATP synthesis and the formation of the permeability transition pore may be responsible for apoptotic cell death already observed in cells treated with BP12.

Changes of the fluidity of mitochondrial membranes induced by the permeability transition.

The dynamic properties of protein and lipid regions of mitochondrial membranes during the permeability transition (PT) process were studied by following the anisotropy changes of hematoporphyrin (HP) and 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. We show that opening of the PT pore is accompanied by a remarkable increase of mitochondrial membrane fluidity which is specifically localized to protein sites, while lipid domains are unaffected. The increased membrane fluidity is not related to the collapse of transmembrane potential that follows the PT, as demonstrated by a comparison between the anisotropy properties of permeabilized mitochondria and impermeable, depolarized organelles. Parameters such as osmotic swelling and temperature, which are shown to affect the mitochondrial membrane dynamics in the absence of permeability transition, cannot alone account for the pore dynamical properties. We suggest that the observed increase in fluidity is mainly due to a conformational change of pore-forming protein(s) during the "assembly" of the PT pore.

Photodynamic action of porphyrin on Ca2+ influx in endoplasmic reticulum: a comparison with mitochondria.

We have studied the distribution properties of haematoporphyrin (HP) and protoporphyrin (PP) in mitochondria and endoplasmic reticulum after isolation from rat liver. The photosensitizing efficiency of porphyrin on the Ca2+ influx function of microsomes has been compared with that obtained on Ca2+ uptake in mitochondria. HP and PP are accumulated in microsomes to a greater extent than in mitochondria, both porphyrins binding to membrane protein sites. The Ca2+ influx functions of mitochondria and microsomes, before and after irradiation in the presence of HP or PP, were studied by following the changes in the free Ca2+ concentration in the medium as revealed by the variations in fluorescence intensity of the Ca2+ indicator Calcium Green-1. For the same amount of incorporated porphyrin, the Ca2+ influx function of microsomes is degraded by irradiation more rapidly than that of mitochondria. The protective effect of dithiothreitol suggests that thiol groups in the Ca2+-transporting enzyme are the preferential targets of the photodynamic effect. These results suggest that intracellular Ca2+ movements are altered primarily by the endoplasmic reticulum rather than by mitochondrial damage, in good agreement with other observations made in porphyrin-loaded irradiated cells.

Effects of photodynamic action on respiration in nonphosphorylating mitochondria.

We have studied the effects of singlet oxygen produced by photodynamic action on respiration in nonphosphorylating mitochondria (state 4). Isolated rat liver mitochondria were incubated with 3 microM hematoporphyrin and irradiated at 365 nm with a fluence rate of 25 W/m2. After short durations of irradiation, state 4 respiration with beta-hydroxybutyrate as substrate increases while respiration with succinate is negligibly affected. When mitochondria have been uncoupled with carbonylcyanide-p-trifluoromethoxyphenyl hydrazone before irradiation, no change occurs in beta-hydroxybutyrate-driven respiration, while succinate-driven respiration strongly decreases. Stimulation of state 4 NADH respiration cannot be explained by slippage of the NADH ubiquinone oxidoreductase because the stoichiometry of the redox pump was found insensitive to photodynamic action. In the light of the metabolite theory for linear enzymatic chains applied to state 4 respiration (Brand et al., Biochem. J. 255, 535-539, 1988), these results suggest that stimulation of NADH respiration is simply due to an increase of membrane leaks which occurs after irradiation. In the case of succinate-driven respiration, a strong inhibition of succinate dehydrogenase activity has been demonstrated after irradiation. It can be suggested that this inhibition introduces a negative control coefficient over state 4 respiration, counterbalancing the effects due to leakage.

Photophysical properties of porphyrin planar aggregates in liposomes.

This paper describes studies of some photophysical properties of non-covalent planar aggregates of hematoporphyrin and protoporphyrin. This porphyrin species has been recently discovered and can be generated in lipid bilayers such as liposomes and inner mitochondrial membranes. The relative weight of this species in different media, as compared to porphyrin monomers and stacked aggregates, has been deduced by fluorescence decay studies. In contrast with what is observed for stacked aggregates, promotion of planar suprastructures can occur both in aqueous and lipid environments. The spectroscopic properties are very similar to those of the corresponding monomers, in particular as regards the shape of the absorption and emission spectra. The fluorescence decay times are generally higher than those of the monomers, and depend on the medium in which the planar aggregates are formed. The photooxidation properties of porphyrin planar aggregates, as revealed by oxygen consumption and histidine photodegradation upon irradiation at 365 nm, were compared to those of the monomers. The extent of the photooxidation process is nearly 20-30% higher in planar aggregates than in the monomers. In contrast, it is well known that cofacial aggregates are photochemically inert and only monomeric species of porphyrin are efficient photosensitizers. The biological relevance of these findings is discussed.

Singlet oxygen produced by photodynamic action causes inactivation of the mitochondrial permeability transition pore.

We have studied the effects of singlet oxygen produced by photodynamic action on the cyclosporin A-sensitive permeability transition (PT) in isolated rat liver mitochondria. Mitochondria were incubated with 3 microM hematoporphyrin and irradiated at 365 nm with a fluence rate of 25 watts/m2. For short durations of irradiation (60 s) the adenine nucleotide translocase was inactivated, but mitochondria retained their ability to form a proton electrochemical gradient and accumulated Ca2+ and Pi at the same rate as non-irradiated controls. Strikingly, however, the oxidative effects of photodynamic action prevented opening of the PT pore which is normally induced by Ca2+ plus Pi or by treatment with diethyl pyrocarbonate (a histidine reagent) or diamide (a thiol oxidant). We show that the most likely targets for photodynamic action are critical histidines that undergo degradation. Irradiated, hematoporphyrin-loaded mitochondria treated with diethyl pyrocarbonate or diamide still undergo the PT when treated with phenylarsine oxide, which reacts with a critical dithiol involved in pore modulation (Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994) J. Biol. Chem. 269, 16638-16642). These data suggest (i) that the dithiol cysteines are not oxidized by photodynamic action, but rather became inaccessible to oxidants; and (ii) that irradiation of hematoporphyrin-loaded mitochondria does not lead to pore denaturation, but rather to site-selective inactivation of discrete pore functional domains.

Induction of mitochondrial dysfunction and apoptosis in HeLa cells by bis-pyridinium oximes, a newly synthesized family of lipophilic biscations.

When tested on HeLa cells, bis-pyridinium oximes (BPO), a family of newly synthesized molecules whose charged pyridinium moieties are linked by a linear polymethylene chain of variable length (N = 3 to 12) have been shown to possess an inhibitory effect on cell growth and finally to provoke cell death. BPO-affected cells displayed reduced mitochondrial oxygen consumption and ATP stores and were blocked in the G1 phase of the cell cycle. Mitochondrial membrane potential, as assayed with the dye 3,3'-diexyloxacarbocyanine iodide [DiOC6(3)], increased in BPO-treated cells with time of exposure. Cell growth inhibition as well mitochondrial dysfunction were observed only with derivatives having a long polymethylene linking chain (N > or = 6). Furthermore, the concentration of the compound eliciting such effects was inversely related to the number of methylene groups in the linking chain. None of the BPO with N = 6 to 12 modified the mitochondrial DNA content, relative to the nuclear DNA content. In BPO (N = 8 and N = 12)-treated cells, chromatin fragmentation and internucleosomal DNA cleavage occurred massively, indicating that the death mode induced by these compounds is apoptosis. The possible pathway of action and the potential pharmacological interest of these compounds are discussed.

Effects of Photofrin photodynamic action on mitochondrial respiration and superoxide radical generation.

Cyanide-resistant respiration increases after irradiation of isolated mitochondria in the presence of Photofrin. This suggests an enhancement of electron leakage which has been evaluated by measuring superoxide radical formation in submitochondrial particles incubated with 6 micrograms/ml Photofrin in the medium and irradiated with increasing doses of light at 365 nm. After a dose of 4.5 kJ/m2 has been delivered, superoxide generation increases by a factor of approximately 2.5 at the level of NADH dehydrogenase and by a factor approximately 1.5 in the cyt bc1 region. These effects have been compared with changes observed in NADH-, succinate- and ascorbate-driven respiration and their implications discussed.

Photosensitizing potencies of the structural analogues of benzoporphyrin derivative in different biological test systems.

Benzoporphyrin derivative (BPD) is a potent photosensitizer in biological systems. There are four structural analogues of BPD. The analogues share the same chromophor, which results in their having almost identical optical spectra, extinction coefficients, and yields of singlet oxygen. Small structural differences affect their photosensitizing potency in various biological systems, and thus make them an interesting tool to study the structure-activity relationship. The ranking of the photosensitizing potency of the analogues differed depending on the test system. The more efficient photosensitization of tumor cell lines by the highly lipophilic monoacids as compared to that by less lipophilic diacids correlated positively with the partition coefficient, and was related to the rate of diffusion into the cells. However, in the assay systems where PDT targets were located in the membrane (red blood cells hemolysis, enveloped vesicular stomatitis virus, isolated mitochondria) there was very little difference in photosensitizing potency of BPD analogues. The results indicate that the evaluation of photosensitizers is affected by the test system and thus for photosensitizers screening purposes, the choice of the test system should be made based on the intended ultimate use.

Temperature-induced changes in fluorescence properties as a probe of porphyrin microenvironment in lipid membranes. 2. The partition of hematoporphyrin and protoporphyrin in mitochondria.

The temperature dependence of hematoporphyrin and protoporphyrin fluorescence quantum yields (phi F) was studied after delivery to whole mitochondria or isolated inner (IMM) and outer (OMM) mitochondrial membranes, obtained from liver of Wistar rats. These studies are very sensitive to variations of the porphyrin lipid environment. Before incorporation, the porphyrins were dissolved in 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4) NaCl/Pi (only hematoporphyrin) or dispersed into liposomes of dipalmitoylphosphoglycerocholine (Pam2GroPCho), sometimes enriched with cholesterol or cardiolipin. Whole mitochondria show higher incorporation capacity of hematoporphyrin and protoporphyrin than isolated IMM and OMM, probably because additional, energy-sensitive transport mechanisms for the porphyrin uptake occur in intact organelles. A small decrease in protoporphyrin uptake is observed in OMM in comparison with IMM; in contrast, the decrease in hematoporphyrin uptake by OMM is rather significant. A comparison between the results obtained with IMM, OMM and whole mitochondria show that both porphyrins, when released to the intact organelles, preferentially localize in the IMM, irrespective of the lipid carrier used. NaCl/Pi-dissolved hematoporphyrin probably interacts with some membrane proteins, due to the similarity of the Arrhenius plots with those obtained for liposome-entrapped human serum albumin/hematoporphyrin complexes which were used as models to mimic hematoporphyrin-membrane protein binding sites. Liposomal hematoporphyrin and protoporphyrin bind to lipid domains. Hematoporphyrin accumulates in specific, localized lipid regions, perhaps in the boundary lipids area surrounding some inner-mitochondrial carriers; protoporphyrin accomodates in more rigid, lipid areas. On these bases, the higher photoactivity of hematoporphyrin, previously observed in mitochondria, in comparison with protoporphyrin, can be easily explained. Formation of linear dimers/aggregates, endowed with higher phi F than that of the monomers, are postulated to occur for both porphyrins only in the inner mitochondrial membrane.

Temperature-induced changes in fluorescence properties as a probe of porphyrin microenvironment in lipid membranes. 1. The partition of hematoporphyrin and protoporphyrin in liposomes.

Temperature-induced fluorescence changes were studied for hematoporphyrin and protoporphyrin, incorporated into liposomes of dipalmitoylphosphoglycerocholine (Pam2GroPCho) or dimiristoylphosphoglycerocholine (Myr2GroPCho). In some cases, cholesterol or cardiolipin were added to the vesicles for better mimicking the lipid composition of biological membranes. The experimental conditions were appropriately chosen in order to reproduce different possible configurations of the porphyrin molecule in lipid membranes: namely, at the polar water/headgroups, headgroups/lipid and lipid/lipid interfaces. A peculiar feature observed in some of the above liposomal systems was the appearance of discontinuities in the Arrhenius plots of the fluorescence quantum yields, with relevant changes of the values of activation energies. These discontinuities were due to an increase of the fluorescence signal in a temperature range corresponding to the transition of the different lipids from the gel-to-liquid crystal state. The observed phenomena are consistent with the formation of non-covalent linear dimers or linear higher aggregates of the porphyrin molecules. The intermolecular contacts required for the formation of these species are favoured by at least three situations: disruption of the ordered lipid structure during the gel-to-liquid crystal phase transition; competition of other polar groups (e.g., the -OH group of cholesterol) with the porphyrin carboxylate groups for the polar phospholipid headgroups; and steric constraints due to overcrowding of porphyrin molecules in a restricted space.

Photodynamic action increases leakage of the mitochondrial electron transport chain.

Rat liver submitochondrial particles were treated with rose bengal or flavin mononucleotide and irradiated with visible or near UV light. In both cases, oxygen consumption is impaired after irradiation while O2-. production increases significantly. This suggests that exogenous or endogenous photosensitizers can induce more electron leakage in the mitochondrial respiratory chain. Photobiological implications in dermatology are discussed.

Interaction of phthalocyanines with lipid membranes: a spectroscopic and functional study on isolated rat liver mitochondria.

Absorption and emission spectroscopic studies on Zn(II)-phthalocyanine (ZnPc) incorporated into unilamellar liposomes of dipalmitoylphosphatidylcholine, sometimes added with cholesterol or cardiolipin, and released to rat liver mitochondria via the three types of liposomal vesicles indicated that: (a) ZnPc predominantly dissolves in all lipid domains of biological membranes with the exception of cardiolipin-containing regions; a partial localization of ZnPc in protein binding sites is also postulated; (b) the spectroscopic properties of ZnPc, although mainly determined by the aggregation state of the dye, are somewhat influenced by the physico-chemical characteristics of the lipid environment; (c) ZnPc-binding lipid domains in mitochondria are mainly localized in the outer membrane; this conclusion is clearly deduced from the trends of Arrhenius plots of the ZnPc fluorescence quantum yield in whole mitochondria and isolated inner or outer membrane in the temperature range -10 degrees C-(+)45 degrees C; (d) the nature of the ZnPc-binding site in mitochondria is not dependent on the chemical composition of the liposome carrier, contrary to what observed for other hydrophobic dyes, such as porphyrins. This has been also confirmed by photosensitization experiments. Actually, illumination of ZnPc-loaded mitochondria by 600-700 nm light causes a decline of the respiratory control ratio, which is essentially dependent on the amount of incorporated photosensitizer, irrespective of the composition of the carrier.

Hematoporphyrin derivative (Photofrin) photodynamic action on Ca2+ transport in monkey kidney cells (CV-1).

After 24 h incubation with Photofrin (PF), photodynamic action has been studied on Ca2+ transport in CV-1 cells. A moderate increase of the cytosolic free Ca2+ concentration [Ca2+]i is observed immediately after a dose of irradiation which yields a survival rate of less than 5% at 48 h. In parallel, studies on digitonin-permeabilized cells indicate that such a treatment inhibits endoplasmic reticulum Ca2+ uptake with few alterations of this process in mitochondria. In contrast, ADP-stimulated respiration is impeded and intracellular ATP level decreases. It is suggested that direct damage to endoplasmic reticulum as well as mitochondrial disturbance are the primary mechanisms responsible for a nontransient elevation of [Ca2+]i preceding cell death.

4-Thiouridine incorporation into the RNA of monkey kidney cells (CV-1) triggers near-UV light long-term inhibition of DNA, RNA and protein synthesis.

Monkey kidney cells (CV-1) grown for 4 h in the presence of 0.1 mM 4-thiouridine (s4Urd) incorporate this photoactivable uridine analog in their RNA. A minor, 5-8%, thiolated RNA fraction can be isolated from bulk RNA by affinity chromatography. This RNA fraction contains 1.5-2.5 s4Urd residues per 100 nucleotides and exhibits a broad chain length distribution ranging from 700 to 7000 nucleotides. It is essentially of nuclear origin and amounts to 30% of the RNA synthesized during exposure of cells to s4Urd. Under the same s4Urd labeling conditions, no thiolated pyrimidine residues have been detected in DNA. Irradiation with 365 nm light (45 kJ/m2) of the cells immediately after s4Urd exposure triggers long-term inhibition of DNA, RNA and protein synthesis accompanied by a linear decline (50% in 2 days) in the total cell mass of cultured cells. In contrast, exposure to s4Urd alone results in moderate but reversible inhibitory effects. The available data suggest that s4Urd-induced photolesions in newly synthesized RNA such as RNA-RNA cross-links as well as RNA-protein bridges are directly involved in impairment of essential cellular functions.

Photosensitization of mitochondria by liposome-bound porphyrins.

We have compared the photodynamic activities of hematoporphyrin (HP) and protoporphyrin (PP) on isolated rat liver mitochondria by measuring the decline of the respiratory control ratio (RCR) after irradiation at 365 nm. Before addition to the respiratory medium, the dyes were dissolved in phosphate-buffered saline (PBS) or incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine (DPPC), sometimes enriched with cholesterol (Chol) or cardiolipin (Card), which are naturally present in mitochondrial membranes. Chol and especially Card strongly increase the porphyrin uptake by mitochondria. In all experimental conditions, PP is taken up by mitochondria to a higher extent than HP. Nevertheless, under conditions giving the same amount of mitochondria-bound dye, HP is a more efficient photosensitizer than PP. As the efficiency of singlet oxygen production has been shown to be equivalent for the two porphyrins in monomeric state, the resulting photobiological effects are explained in terms of different localization of HP and PP in the mitochondrial membranes. In particular, HP preferentially localizes in the protein-rich polar domains of the inner mitochondrial membrane, whereas PP dissolves in the lipid regions of the membranes.

Cytotoxic and photodynamic effects of Photofrin on sensitive and multi-drug-resistant Friend leukaemia cells.

To study cross-resistance to Photofrin (PF) photosensitization, a Friend leukaemia cell line (ADM-RFLC) with a high level of multi-drug resistance (MDR) and the parental sensitive cell line (FLC) have been used. PF uptake measured by HPLC shows a similar intracellular drug accumulation in both cell lines. The ID50s for cell growth inhibition by PF are also similar after exposure in the dark in the two cell lines, while after illumination they are slightly lower in ADM-RFLC than in FLC cells. Moreover, verapamil, known to reverse the MDR phenotype induced by P-glycoprotein over-expression (the drug efflux mechanism), affects equally ADM-RFLC and FLC cells sensitivity to PF. In addition, photodynamic treatment with PF did not reverse the resistance to rhodamine 123 and aclarubicin, but partly reverses resistance of ADM-RFLC cells to antitubulin drugs such as vinblastine or vincristine. These latter results could have clinical application in the treatment of tumours expressing the MDR phenotype.

Effects of rhodamine 123 in the dark and after irradiation on mitochondrial energy metabolism.

Isolated rat liver mitochondria have been used to study the mechanism of toxicity of Rhodamine 123 (Rho 123) in the dark and after irradiation with visible light. We report an inhibition of adenosine 5'-diphosphate phosphorylation which is increased after illumination. In the dark, the first steps of the phosphorylation process (i.e. the entry of substrates into the matrix, the electron transport to oxygen and the creation of the proton gradient) as well as ATPase activity are not significantly perturbed at Rho 123 concentration below 10 micrograms/mL. In contrast, the movements of the phosphate compounds are drastically impaired. Irradiation strengthens the detrimental effects in an oxygen dependent process. The nature of the noxious transient species is not clearly established, but it is suggested that singlet oxygen could be responsible for the observed damage.