Effect of the lipophilic/hydrophilic character of cationic triarylmethane dyes on their selective phototoxicity toward tumor cells

The observation that enhanced mitochondrial transmembrane potential is a prevalent tumor cell phenotype has provided the conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemo- and photochemotherapy of neoplastic diseases. Because the plasma transmembrane potential is negative on the inner side of the cell and the mitochondrial transmembrane potential is negative on the inner side of this organelle, extensively conjugated cationic molecules (dyes) displaying appropriate structural features are driven electrophoretically through these membranes and tend to accumulate inside energized mitochondria. As a result of the higher mitochondrial transmembrane potential typical of tumor cells, a number of cationic dyes preferentially accrue and are retained for longer periods in the mitochondria of these cells compared to normal cells. This differential in both drug loading and retention brings about the opportunity to attack and destroy tumor cells with a high degree of selectivity. Only a small subset of the cationic dyes known to accumulate in energized mitochondria mediate the destruction of tumor cells with a high degree of selectivity, and the lack of a reliable model to describe the structural determinants of this tumor specificity has prevented mitochondrial targeting from becoming a more reliable therapeutic strategy. We describe here a systematic study of how the molecular structure of closely related cationic triarylmethanes affects the selectivity with which these dyes mediate the photochemical destruction of tumor cells. Based on our observations of how the lipophilic/hydrophilic character of these dyes affects tumor selectivity, we propose a simple model to assist in the design of new drugs tailored specifically for imaging and selective destruction of neoplastic tissue via mitochondrial targeting.     

Effect of the lipophilic/hydrophilic character of cationic triarylmethane dyes on their selective phototoxicity toward tumor cells.

The observation that enhanced mitochondrial transmembrane potential is a prevalent tumor cell phenotype has provided the conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemo- and photochemotherapy of neoplastic diseases. Because the plasma transmembrane potential is negative on the inner side of the cell and the mitochondrial transmembrane potential is negative on the inner side of this organelle, extensively conjugated cationic molecules (dyes) displaying appropriate structural features are driven electrophoretically through these membranes and tend to accumulate inside energized mitochondria. As a result of the higher mitochondrial transmembrane potential typical of tumor cells, a number of cationic dyes preferentially accrue and are retained for longer periods in the mitochondria of these cells compared to normal cells. This differential in both drug loading and retention brings about the opportunity to attack and destroy tumor cells with a high degree of selectivity. Only a small subset of the cationic dyes known to accumulate in energized mitochondria mediate the destruction of tumor cells with a high degree of selectivity, and the lack of a reliable model to describe the structural determinants of this tumor specificity has prevented mitochondrial targeting from becoming a more reliable therapeutic strategy. We describe here a systematic study of how the molecular structure of closely related cationic triarylmethanes affects the selectivity with which these dyes mediate the photochemical destruction of tumor cells. Based on our observations of how the lipophilic/hydrophilic character of these dyes affects tumor selectivity, we propose a simple model to assist in the design of new drugs tailored specifically for imaging and selective destruction of neoplastic tissue via mitochondrial targeting.     

lmmunocytochemical Detection of Bromodeoxyuridine in Tissues of the Toad Bufo arenarum Hensel

Abstract

The concept of mitochondrial targeting for chemo- and photochemotherapy of neoplastic diseases has its origin in the observation that enhanced mitochondrial transmembrane potential is a common tumor cell phenotype. As a result of this enhanced transmembrane potential, a number of cationic dyes accumulate in larger amounts and are retained for longer periods in the mitochondria of tumor cells than in normal cells. Only a relatively small number of (photo)toxic dyes known to localize in energized cell mitochondria are capable of inducing the destruction of tumor cells with desirable degrees of selectivity, however. We investigated how lipophilic character may affect the degree of specificity with which cationic dyes localize in energized cell mitochondria and how mitochondrial specificity may affect tumor cell selectivity. To this end, we used fluorescence microscopy to characterize the subcellular localization of ethyl violet and seven analogs of the prototypical mitochondria-specific dye, rhodamine 123. All cationic rhodamines studied here (?0.62 < log D_ow < 1.60, where D_ow represents the n-octanol/water distribution coefficient) were found to show considerable mitochondrial specificity, while the more lipophilic ethyl violet (log D_ow = 2.37) did not. Ethyl violet was found to localize not only in mitochondria, but also in lysosomes. We also compared the degree of selective tumor cell killing induced by ethyl violet and two phototoxic rhodamines, i.e., the dibromo derivatives of rhodamine 123 and its n-octyl ester analog. While ethyl violet induces the destruction of human uterine sarcoma (MES-SA) cells and normal green monkey kidney cells (CV-1) with comparable efficiency, the mitochondria-specific dibromorhodamines were found to induce the destruction of MES-SA cells with considerable selectivity. Our findings are consistent with the premise that mitochondrial localization per se does not provide successful selective tumor cell killing using mitochondrial targeting. Our results reinforce the hypothesis that while most cationic dyes can be expected to localize at least to some extent in energized cell mitochondria, only those showing virtually absolute mitochondrial specificity can actually mediate the destruction of tumor cells with desirable selectivity. These findings also support the hypothesis that the probability of success of mitochondrial targeting in photochemotherapy of neoplastic diseases is bound to be higher when the D_ow associated with the drug candidate falls within approximately two orders of magnitude of that of rhodamine 123.     

A predictive model for the selective accumulation of chemicals in tumor cells

Cationic lipophilic dyes can accumulate in mitochondria, and especially in mitochondria of tumor cells. We investigated the chemical properties and the processes allowing selective uptake into tumor cells using the Fick–Nernst–Planck equation. The model simulates uptake into cytoplasm and mitochondria and is valid for neutral molecules and ions, and thus also for weak electrolytes. The differential equation system was analytically solved for the steady-state and the dynamic case. The parameterization was for a generic human cell, with a 60 mV more negative potential at the inner mitochondrial membrane of generic tumor cells. The chemical input data were the lipophilicity (logK_OW), the acid/base dissociation constant (pK_a) and the electric charge (z). Accumulation in mitochondria occurred for polar acids with pK_a between 5 and 9 owing to the ion trap, and for lipophilic bases with pK_a>11 or permanent cations owing to electrical attraction. Selective accumulation in tumor cells was found for monovalent cations or strong bases with logK_OW of the cation between –2 and 2, with the optimum near 0. The results are in agreement with experimental results for rhodamine 123, a series of cationic triarylmethane dyes, F16 and MKT-077, an anticancer drug targeting tumor mitochondria.     

Interaction of electron leak and proton leak in respiratory chain of mitochondria——Proton leak induced by superoxide from an electron leak pathway of univalent reduction of oxygen

By incubating the isolated rat myocardial mitochondria with xanthine-xanthine oxidase, anexogenous superoxide (O2) generating system, and by ischemia-reperfusion procedure of isolated rat heart as an endogenous O2 generating system, it was found that both sources of O2 showed the same injurious effects on mitochondrial function resulting in (i) increasing proton leak rate, lowering proton pumping activity and Ht/2e ratio of respiratory chain, and (ii) decreasing transmembrane potential of energized mitochondria] inner membrane by succinate oxidation. The injurious effects of O2 on these mitochondrial bioenergitical parameters mentioned above exhibited a dosage- or reaction time-dependent mode. (X has no effects on the electron transfer activity and transmembrane potential of nonenergized mitochondria. Being a superoxide scavenger, 3, 4-dihydroxylphenyl lactate showed obvious protection effects against damage of both exogenous superoxide sources from xanthine-xanthine oxidase system and endogenous Or sou     

On measurement of mitochondrial transmembrane potential with fluorescent probes

It is commonly thought that rhodamine, cyanine, and some other fluorescent dyes are specific potential-dependent ones and that they allow quantitatively measuring the transmembrane potential in mitochondria and cells. However, a critical analysis of the experimental data shows that this statement is only a supposition. In reality, widely used fluorescent probes, such as merocyanine 540, Dis-C3-(5), safranin O, or 8-anilino-1-naphthalene sulfonate, poorly bind to the native mitochondria and weakly react to their energization or uncoupling. It can be concluded that calculations of the magnitude of the transmembrane potential of the inner mitochondrial membrane in response to addition of succinate, ATP, or dinitrophenol from the change in fluorescence of these probes are incorrect.     

64Cu-labeled phosphonium cations as PET radiotracers for tumor imaging

Alteration in mitochondrial transmembrane potential (ΔΨ(m)) is an important characteristic of cancer. The observation that the enhanced negative mitochondrial potential is prevalent in tumor cell phenotype provides a conceptual basis for development of mitochondrion-targeting therapeutic drugs and molecular imaging probes. Since plasma and mitochondrial potentials are negative, many delocalized organic cations, such as rhodamine-123 and (3)H-tetraphenylphosphonium, are electrophoretically driven through these membranes, and able to localize in the energized mitochondria of tumor cells. Cationic radiotracers, such as (99m)Tc-Sestamibi and (99m)Tc-Tetrofosmin, have been clinically used for diagnosis of cancer by single photon emission computed tomography (SPECT) and noninvasive monitoring of the multidrug resistance (MDR) transport function in tumors of different origin. However, their diagnostic and prognostic values are often limited due to their insufficient tumor localization (low radiotracer tumor uptake) and high radioactivity accumulation in the chest and abdominal regions (low tumor selectivity). In contrast, the (64)Cu-labeled phosphonium cations represent a new class of PET (positron emission tomography) radiotracers with good tumor uptake and high tumor selectivity. This review article will focus on our recent experiences in evaluation of (64)Cu-labeled phosphonium cations as potential PET radiotracers. The main objective is to illustrate the impact of radiometal chelate on physical, chemical, and biological properties of (64)Cu radiotracers. It will also discuss some important issues related to their tumor selectivity and possible tumor localization mechanism.     

Giant mitochondria do not fuse and exchange their contents with normal mitochondria

Giant mitochondria accumulate within aged or diseased postmitotic cells as a consequence of insufficient autophagy, which is normally responsible for mitochondrial degradation. We report that giant mitochondria accumulating in cultured rat myoblasts due to inhibition of autophagy have low inner membrane potential and do not fuse with each other or with normal mitochondria. In addition to the low inner mitochondrial membrane potential in giant mitochondria, the quantity of the OPA1 mitochondrial fusion protein in these mitochondria was low, but the abundance of mitofusin-2 (Mfn2) remained unchanged. The combination of these factors may explain the lack of mitochondrial fusion in giant mitochondria and imply that the dysfunctional giant mitochondria cannot restore their function by fusing and exchanging their contents with fully functional mitochondria. These findings have important implications for understanding the mechanisms of accumulation of age-related mitochondrial damage in postmitotic cells.     

Fluorescent cationic probes of mitochondria. Metrics and mechanism of interaction.

Mitochondria strongly accumulate amphiphilic cations. We report here a study of the association of respiring rat liver mitochondria with several fluorescent cationic dyes from differing structural classes. Using gravimetric and fluorometric analysis of dye partition, we find that dyes and mitochondria interact in three ways: (a) uptake with fluorescence quenching, (b) uptake without change in fluorescence intensity, and (c) lack of uptake. For dyes that quench upon uptake, the extent of quenching correlates with the degree of aggregation of the dye to dimers, as predicted by theory (Tomov, T.C. 1986. J. Biochem. Biophys. Methods. 13:29-38). Also predicted is the relationship observed between quenching and the mitochondria concentration when constant dye is titrated with mitochondria. Not predicted is the relationship observed between quenching and dye concentration when constant mitochondria are titrated with dye. Because a limit to dye uptake exists, in this case, the degree of quenching decreases as dye is added. A Langmuir isotherm analysis gives phenomenological parameters that predict quenching when it is observed as a function of dye concentration. By allowing for a decrease in membrane potential, caused by incorporation of cationic dye into the lipid bilayer, a modification of the Tomov theory predicts the dye titration data. We present a model of cationic dye-mitochondria interaction and discuss the use of these as probes of mitochondrial membrane potential.     

Does the energy state of mitochondria influence the surface potential of the inner mitochondrial membrane? A critical appraisal

Binding of 8-anilino-1-naphthalene sulphonate (ANS) to rat liver mitochondria and submitochondrial inside-out particles was measured under energized and de-energized conditions. In mitochondria, energization/de-energization changed the binding capacity for ANS extrapolated for its infinitely high concentration, whereas the apparent Kd value remained unchanged. In submitochondrial particles apparent Kd was changed but the extrapolated maximum binding was not altered. These results are compatible with theoretical considerations assuming a free permeability of mitochondrial membranes to ANS and its distribution according to the transmembrane potential. The spin-labelled cationic amphiphile, 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6-tetramethyl piperidine bromide (CAT12), was trapped by de-energized mitochondria in such a way that about half of the bound probe became inaccessible to reduction by externally added ascorbate. This inaccessible fraction was increased by energization. This indicates that this cationic probe can penetrate through the inner mitochondrial membrane. De-energization produced a parallel shift of the Lineweaver-Burk plots for the oxidation of external ferrocytochrome c by mitoplasts and of succinate by submitochondrial particles. A similar shift was obtained by a partial inhibition of succinate oxidation by antimycin A. Thus, the observed changes of the kinetics of the two membrane-bound enzyme systems on de-energization can be interpreted as reflecting changes of the control points of mitochondrial respiration rather than changes of the surface potential. It is concluded that neither the fluorescent probe ANS, the spin-labelled amphiphilic cation CAT12, nor the kinetics of some respiratory enzyme systems provide a sufficient proof for changes of the surface potential of the inner mitochondrial membrane upon energization.     

Pontentiometric cyanine dyes are sensitive probes for mitochondria in intact plant cells : kinetin enhances mitochondrial fluorescence

Selected fluorescent dyes were tested for uptake by mitochrondria in intact cells of barley, maize, and onion. The cationic cyanine dye 3,3′-diheptyloxacarbocyanine iodide [DiOC₇(3)] accumulated in mitochondria within 15 to 30 minutes without appreciable staining of other protoplasmic constituents. The number, shape, and movement of the fluorescent mitochondria could be seen readily, and the fluorescence intensity of the mitochondria could be monitored with a microscope photometer. Fluorescence was eliminated in 1 to 5 minutes by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) indicating that maintenance of dye concentration was dependent on the inside-negative transmembrane potential maintained by functional mitochondria. Fluorescence of prestained mitochondria was enhanced within 5 to 10 minutes after addition of 0.1 millimolar kinetin to cells. The fluorescence in kinetintreated cells was dissipated by CCCP. These results suggest that kinetin interacted with respiratory processes resulting in higher potential across the mitochondrial membrane.     

A synthetic signal peptide blocks import of precursor proteins destined for the mitochondrial inner membrane or matrix

A peptide corresponding to amino acids 1-27 of preornithine carbamyltransferase (pOCT) has been chemically synthesized. When added to energized mitochondria in vitro, 20 microM of the peptide, designated pO(1-27), resulted in a collapse of the electrochemical potential across the mitochondrial inner membrane. This effect on transmembrane potential was not observed, however, when pO(1-27) was added to energized mitochondria under conditions that support in vitro import of precursor proteins (i.e. in the presence of reticulocyte lysate). The latter finding, therefore, made possible an examination of the ability of pO(1-27) to block import of homologous and heterologous proteins into the organelle. At 5-10 microM, pO(1-27) prevented import of pOCT in vitro; inhibition was overcome by increasing the concentration of pOCT. In contrast, pO(16-27), a peptide corresponding to amino acids 16-27 of pOCT and exhibiting a charge:mass ratio similar to pO(1-27) had no such inhibitory effect. pO(1-27) blocked import of other unrelated precursor proteins destined either for the mitochondrial matrix (pre-malate dehydrogenase and a hybrid protein containing the signal sequence of pre-carbamyl phosphate synthetase) or for the mitochondrial inner membrane (pre-thermogenin).     

Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB.     

Berberine derivatives as cationic fluorescent probes for the investigation of the energized state of mitochondria

The interaction of rat liver and bovine heart mitochondria with a series of fluorescent, cationic berberine derivatives varying in the length of alkyl chain has been investigated. An increase in the hydrophobicity of the derivative was accompanied by a larger value of the partition coefficient and by binding to a more hydrophobic region of the inner mitochondrial membrane. It was found that berberines could be used as sensitive indicators of processes which take place on the outer surface of the mitochondrial membrane; the greatest (15-fold) increase in fluorescence was obtained with 13-methylberberine in the energized state of mitochondria. The fluorescence increase was due to the increase in fluorescence quantum yield although a small increase in the amount of bound derivative could also be detected upon energization. The fluorescence was linearly dependent on the magnitude of the membrane potential. In parallel with an observed fluorescence enhancement a considerable decrease in rotational mobility was found. We suggest that berberines move in the inner membrane according to the polarity of the membrane potential; consequently, deeper immersion in the less polar region in the energized state brings about a larger fluorescence increase. More hydrophobic derivatives inhibited NAD-linked respiration in rat liver mitochondria but exerted no effect on succinate oxidation up to 10 μM concentration.     

Photoaffinity labelling with fluorescence detection

A micromethod was developed for investigating the interactions between fluorescent dyes and cellular proteins. The lipophilic cationic dye APMC (azopentylmethylcarbocyanine) contains a photosensitive diazirine ring and is suitable for photoaffinity labelling. By combining photoaffinity labelling of cultured cells, micro-gel electrophoresis and detection of the fluorescence with a microfluorimeter, we established a highly sensitive and rapid procedure to identify APMC labelled proteins. Cells which had been incubated for 10 min with 10^–8 M APMC could be analysed for APMC binding without difficulty. Under our experimental conditions this corresponds to about 0.2 nmol APMC per mg protein. The lipophilic APMC specifically stains the mitochondria in living HeLa and LM cells. The fluorescing mitochondria can be easily detected under a fluorescence microscope. By photoaffinity labelling we were able to show that at low dye concentrations APMC preferentially marks four proteins with apparent molecular masses of 31, 40, 66, and 74 kDa. In order to establish that these are mitochondrial proteins, we isolated and analysed the mitochondria from incubated HeLa and LM cells; again, the same four proteins were detected. They are most probably proteins of the inner mitochondrial membranes, which accumulate the lipophilic APMC cations.     

EphrinA I-targeted nanoshells for photothermal ablation of prostate cancer cells

Gold-coated silica nanoshells are a class of nanoparticles that can be designed to possess strong absorption of light in the near infrared (NIR) wavelength region. When injected intravenously, these nanoshells have been shown to accumulate in tumors and subsequently mediate photothermal treatment, leading to tumor regression. In this work, we sought to improve their specificity by targeting them to prostate tumor cells. We report selective targeting of PC-3 cells with nanoshells conjugated to ephrinA I, a ligand for EphA2 receptor that is overexpressed on PC-3 cells. We demonstrate selective photo-thermal destruction of these cells upon application of the NIR laser.     

Protein degradation within mitochondria: versatile activities of AAA proteases and other peptidases

Cell survival depends on essential processes in mitochondria. Various proteases within these organelles regulate mitochondrial biogenesis and ensure the complete degradation of excess or damaged proteins. Many of these proteases are highly conserved and ubiquitous in eukaryotic cells. They can be assigned to three functional classes: processing peptidases, which cleave off mitochondrial targeting sequences of nuclearly encoded proteins and process mitochondrial proteins with regulatory functions; ATP-dependent proteases, which either act as processing peptidases with regulatory functions or as quality-control enzymes degrading non-native polypeptides to peptides; and oligopeptidases, which degrade these peptides and mitochondrial targeting sequences to amino acids. Disturbances of protein degradation within mitochondria cause severe phenotypes in various organisms and can lead to the induction of apoptotic programmes and cell-specific neurodegeneration in mammals. After an overview of the proteolytic system of mitochondria, we will focus on versatile functions of ATP-dependent AAA proteases in the inner membrane. These conserved proteolytic machines conduct protein quality surveillance of mitochondrial inner membrane proteins, mediate vectorial protein dislocation from membranes, and, acting as processing enzymes, control ribosome assembly, mitochondrial protein synthesis, and mitochondrial fusion. Implications of these functions for cell-specific axonal degeneration in hereditary spastic paraplegia will be discussed.     

大鼠急性应激时肝线粒体质子跨膜转运活性的调控

大鼠烫伤早期(烫伤后30min),肝线粒体质子和电子传递速度均加快,线粒体能化态跨膜电位降低(均以琥珀酸为底物),线粒体膜脂流动性降低。皮下注射去甲肾上腺素后也有上述现象发生。推测急性应激通过儿茶酚胺类作用于肝细胞,导致线粒体内膜有序性增强所致。     

F1-catalysed ATP hydrolysis is required for mitochondrial biogenesis in Saccharomyces cerevisiae growing under conditions where it cannot respire

Mutant strains of yeast Saccharomyces cerevisiae lacking a functional F1-ATPase were found to grow very poorly under anaerobic conditions. A single amino acid replacement (K222 > E222) that locally disrupts the adenine nucleotide catalytic site in the beta-F1 subunit was sufficient to compromise anaerobic growth. This mutation also affected growth in aerated conditions when ethidium bromide (an intercalating agent impairing mtDNA propagation) or antimycin (an inhibitor of respiration) was included in the medium. F1-deficient cells forced to grow in oxygen-limited conditions were shown to lose their mtDNA completely and to accumulate Hsp60p mainly under its precursor form. Fluorescence microscopy analyses with a modified GFP containing a mitochondrial targeting presequence revealed that aerobically growing F1-deficient cells stopped importing the GFP when antimycin was added to the medium. Finally, after total inactivation of the catalytic alpha3beta3 subcomplex of F1, mitochondria could no longer be energized by externally added ATP because of either a block in assembly or local disruption of the adenine nucleotide processing site. Altogether these data strengthen the notion that in the absence of respiration, and whether the proton translocating domain (F0) of complex V is present or not, F1-catalysed hydrolysis of ATP is essential for the occurrence of vital cellular processes depending on the maintenance of an electrochemical potential across the mitochondrial inner membrane.     

Mitochondrial Effects of Triarylmethane Dyes

The mitochondrial effects of submicromolar concentrations of six triarylmethane dyes, withpotential applications in antioncotic photodynamic therapy, were studied. All dyes promotedan inhibition of glutamate or succinate-supported respiration in uncoupled mitochondria, in amanner stimulated photodynamically. No inhibition of N,N,N,N-tetramethyl-p-phenylenediamine(TMPD) supported respiration was observed, indicating that these dyes do not affectmitochondrial complex IV. When mitochondria were energized with TMPD in the absence ofan uncoupler, treatment with victoria blue R, B, or BO, promoted a dissipation of mitochondrialmembrane potential and increase of respiratory rates, compatible with mitochondrialuncoupling. This effect was observed even in the dark, and was not prevented by EGTA, Mg²⁺ orcyclosporin A, suggesting that it is promoted by a direct effect of the dye on inner mitochondrialmembrane permeability to protons. Indeed, victoria blue R, B, and BO promoted swellingof valinomycin-treated mitochondria incubated in a hyposmotic K⁺-acetate-based medium,confirming that these dyes act as classic protonophores such as FCCP. On the other hand, ethylviolet, crystal violet, and malachite green promoted a dissipation of mitochondrial membranepotential, accompanied by mitochondrial swelling, which was prevented by EGTA, Mg²⁺, andcyclosporin A, demonstrating that these drugs induce mitochondrial permeability transition.This mitochondrial permeabilization was followed by respiratory inhibition, attributable tocytochrome c release, and was caused by the oxidation of NAD(P)H promoted by these drugs.