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Novel Mycotoxin from Acremonium exuWiarum Is a Powerful Inhibitor of the Mitochondrial Respiratory Chain Complex III Alexey G. Kruglov,†,‡ Maria A. Andersson,† Raimo Mikkola,† Merja Roivainen,§ Laszlo Kredics,| Nils-Erik L. Saris,† and Mirja S. Salkinoja-Salonen*,† Department of Applied Chemistry and Microbiology, UniVersity of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland, Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Institutskaya 3, Pushchino, Moscow Region, 142290 Russia, Department of Viral Diseases and Immunology, National Public Health Institute, Mannerheimintie 166, FI-00300 Helsinki, Finland, and Department of Microbiology, Faculty of Science and Informatics, UniVersity of Szeged, Kozep fasor 52, H-6726 Szeged, Hungary ReceiVed August 21, 2008
A novel mycotoxin named acrebol, consisting of two closely similar peptaibols (1726 and 1740 Da), was isolated from an indoor strain of the mitosporic ascomycete fungus Acremonium exuViarum. This paper describes the unique mitochondrial toxicity of acrebol, not earlier described for any peptaibol. Acrebol inhibited complex III of the respiratory chain of isolated rat liver mitochondria (1 mg of protein mL-1) with an IC50 of ∼80 ng mL-1 (50 nM) after a short preincubation, and 350 ng mL-1 caused immediate and complete inhibition. Acrebol thus is a complex III inhibitor almost as potent as antimycin A and myxothiazol but completely different in structure. Similarly to myxothiazol but in contrast to antimycin A, acrebol decreased the level of mitochondrial superoxide anion detectable by chemiluminescent probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one. Unlike other peptaibols, acrebol in toxic concentrations did not increase the ionic and solute permeability of membranes of isolated rat liver mitochondria, did not induce disturbance of the ionic homeostasis or the osmotic balance of mitochondria, and did not release apoptogenic proteins like cytochrome c from the intermembrane space of mitochondria. In boar spermatozoa, acrebol inhibited the respiratory chain and caused ATP depletion by activation of the oligomycin-sensitive F0F1-ATPase, which resulted in the inhibition of the progressive movement. In mouse insulinoma MIN-6 cells, whose energy supply solely depends on oxidative phosphorylation, acrebol induced necrosis-like death. The pathophysiological relevance of these findings is discussed. Introduction Filamentous fungi produce numerous bioactive substances, with fungicidal, bactericidal, or antiviral properties. We looked for mycotoxins (i.e., for metabolites toxic to mammalian cells) in fungi colonizing indoor material in a water-damaged residential building where the occupants suffered from serious, building-associated ill health. Boar spermatozoa have been successfully used as a tool for searching toxic peptaibols from fungi (1) and were therefore employed as a tool in this study. The sperm possess an inefficient aerobic energy metabolism (2, 3) and are particularly sensitive to substances damaging mitochondria (4). A filamentous fungus, identified as Acremonium exuViarum, was found to produce substances that were toxic toward the spermatozoa. The toxic molecules from A. exuViarum were identified as novel peptaibols, named acrebol (5). Acrebol consists of two closely similar peptaibols, with molecular masses of 1726 and 1740 Da, consisting of the peptide acetyl-Phe1-Iva2/Val2-Gln3-Aib4-Ile5-Thr6-Leu7-Aib8/Val8Pro9-Aib10-Gln11-Pro12-Aib13-(X-14-X15-X16)-Ser17OH. Acrebol caused irreversible loss of the progressive movement * To whom correspondence should be addressed. Tel: +358(0)40 5739049. Fax: 358(0)9 19159301. E-mail: mirja.salkinoja-salonen@ helsinki.fi. † University of Helsinki. ‡ Russian Academy of Sciences. § National Public Health Institute. | University of Szeged.
of boar spermatozoa at an exposure concentration of 250 ng mL-1, but the exposed cells did not take up propidium iodide, indicating that they were live in spite of being immotile (5). We hypothesized that the subcellular targets of acrebol toxicity may have been the mitochondria, on which sperm motility is critically dependent. Peptaibols are defined as linear amphipatic, R-aminoisobutyric acid (Aib)1-rich peptides, nonribosomally synthesized by peptide synthetases of fungi imperfecti. Most of the peptaibols described in the literature are products of members of the genera Trichoderma, Acremonium, Paecilomyces, and Emericellopsis (for recent reviews, see refs 6-10). The biological effects of peptaibols are believed to be determined by their ability to incorporate into planar lipid bilayer membranes (11). Upon the association into oligomeric assemblies, the peptaibols may form ion channels as exemplified by trichosporin-Bs (12), trichocellins (13), hypelcins (14), antiamoebin (15, 16), and zervamicins (17) or pores for low molecular weight solutes, for example, 1 Abbreviations: Aib, R-aminoisobutyric acid; BSA, bovine serum albumin; cyt c, cytochrome c; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; FCCP, carbonyl cyanidep-(trifluoromethoxy)phenyl-hydrazone; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; Hyp, hydroxyproline; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MCLA, 3,7-dihydro-2-methyl-6-(4methoxyphenyl)imidazol[1,2-a]pyrazine-3-one; Qi-center, inner quinone binding center of complex III; Qo-center, outer quinone binding center of complex III; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine; TPP+, tetraphenylphosphonium; ∆Ψm, mitochondrial transmembrane potential.
10.1021/tx800317z CCC: $40.75 2009 American Chemical Society Published on Web 02/05/2009
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alamethicin (6, 18, 19), trichovirin (20), chrysospermin (21), and hypelcin A (22, 23). This leads to an increase in the conductivity of membranes toward ions and solutes, a disturbance of ionic homeostasis, a loss of the osmotic balance, and cell death. As the bioactivities of all peptaibols reported so far were due to the disturbance of the ionic and osmotic balances in cells and mitochondria (1, 19, 20, 23), we hypothesized that the inhibition of sperm motility by acrebol was connected to its ability to form ion channels or pores for low molecular weight solutes. Against the expectations, the results, described in this paper, showed that the toxic action of acrebol was connected to inhibition of the mitochondrial respiratory complex III and not to formation of any solute-permeating channels or pores. We describe here the mitochondriotoxic effects of acrebol. We used as tools isolated rat liver mitochondria, sperm cells, and mouse insulinoma (MIN-6) cells, of which the energy metabolism is known to rely on mitochondria (24-26).
Experimental Procedures Preparation and Identification of Acrebol. The fungus A. exuViarum strain BMB4 (DSM 21752) was isolated from the moisture-damaged material of a residential building where the occupants suffered from building-related ill health symptoms. Hyphal biomass of the strain BMB4 was extracted and fractionated by RP-HPLC as described by Andersson et al. (5). The fractions for further analysis were selected, and their purification was guided by motility inhibition of boar spermatozoa. Separation was done by isocratic elution using 20% 0.1% formic acid (A) and 80% methanol (B) for 20 min, continuing with a gradient to 100% B for 30 min, and maintaining 100% B for 40 min at a flow rate of 1 mL min-1. For detection, absorbance at wavelengths of 215, 240, 254, and 280 nm was used. Fractions were collected once a minute. The concentrations of peptaibols in the methanol extracts were determined with HPLC using the peptaibol alamethicin (Sigma, A4665, St. Louis, MO) as a reference compound. The acrebols were identified based on their mass ions obtained by electrospray ionization ion trap mass spectrometry (MSD-Trap-XCT_plus ion trap mass spectrometer equipped with Agilent source and Agilent 1100 series LC; Agilent Technologies, Wilmington, DE). Acrebol A gave a single charged adduct [M + Na]+at m/z 1748.9 (highest abundance) and a double charged [M + Na]2+ adduct at m/z 886.1, and acrebol B gave single charged and double charged [M + Na] adducts at m/z 1762.9 and m/z 893.1, respectively. Isolation and Purification of Rat Liver Mitochondria. All animal manipulations before the beginning isolation of livers were performed by the staff of the Animal Department of the Biocenter, University of Helsinki, in accordance with ethical standards as formulated in the Helsinki Declaration of 1975 (revised 1983) and national requirements for the care and use of laboratory animals. The rats were killed by cutting the neck after anesthetization with CO2. Rat liver mitochondria were isolated according to a standard differential centrifugation procedure (27) modified as described by Teplova et al. (28). The standard homogenization medium contained 220 mM mannitol, 70 mM sucrose, 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) (pH adjusted to 7.4 with Trizma Base), 1 mM ethylene glycol-bis(2-aminoethylether)N,N,N′,N′-tetraacetic acid (EGTA), and 0.5% bovine serum albumin (BSA). The final pellets were resuspended in this medium to yield 80-90 mg protein mL-1, assayed by the biuret method (29) with BSA as the standard. The purity of the mitochondria obtained by this method showed that the contamination with microsomes was insignificant (30); further purification was not reasonable due to an increase of mechanical damage to the mitochondria. Activity measurements, unless otherwise indicated, were performed at 30 °C with 1 mg of mitochondrial protein mL-1, using standard KCl-
KrugloV et al. based medium (125 mM KCl, 20 mM mannitol, 10 mM HEPES, and 2 mM KH2PO4) supplemented with 5 mM glutamate and 5 mM malate. Recording the Permeabilization of Mitochondrial Membranes for Ions and Solutes. Swelling of the isolated mitochondria was recorded as a decrease in A540 in standard KCl-based medium and in media where KCl was replaced by KNO3 (125 mM) or by NaCl (125 mM) and KH2PO4 replaced by NaH2PO4 (2 mM). The permeability of membranes for low molecular weight solutes was recorded in inorganic phosphate-free sucrose/mannitol-based medium (60 mM sucrose, 200 mM mannitol, and 10 mM HEPES). The mitochondrial transmembrane potential (∆Ψm) was assessed by measuring the distribution of 1 µM tetraphenylphosphonium (TPP+) between the mitochondria and the solution using a TPP+selective electrode, according to the equation ∆Ψm ) lg ([TPP+]in/ [TPP+]out) (28, 31). Changes in the permeability of the mitochondrial membranes for K+ and Ca2+ in the presence of acrebol were assessed with the use of the respective ion-selective electrodes. To prevent a potential-dependent leak of ions from the deenergized mitochondria, 2 mM MgATP was added to the medium. A Record 4 system was used for the data collection (Institute of Theoretical and Experimental Biophysics RAS, Russia). Measurements of the Rate of Oxygen Consumption by Isolated Rat Liver Mitochondria. The mitochondrial respiration was polarographically recorded using a Clark type oxygen electrode, Oxygraph PS-Peltier PAAR, and the software of DatLab (OROBOROS, Innsbruck, Austria). For simultaneous recording of several parameters, the oxygen electrode was connected to Record 4. Recording the Changes of the NAD(P)H Redox State in the Mitochondria. The changes were recorded using Victor3 multiplate reader (Perkin-Elmer, United States) and 96 well plates at 360 (excitation) and 480 nm (emission). The incubation mixture contained 0.25-0.3 mg of mitochondrial protein per mL. Measurement of Cytochrome c (cyt c) Release from the Mitochondria. Rat liver mitochondria (2 mg mL-1 in a 6 mL chamber) were incubated in standard KCl-based medium supplemented with respiratory substrates with continuous control of the respiration and the ∆Ψm and parallel control of swelling. Swelling and/or a drop in ∆Ψm was initiated by addition of the indicated amounts of Ca2+, alamethicin, valinomycin, and inhibitors of the respiratory chain. After a 10 min incubation with stirring, the mitochondria were sedimented by centrifugation, and the differential absorption spectra of the supernatant (reduced with dithionite/ oxidized) were recorded in broad cuvettes [path (l) ) 3 cm]. The extinction coefficient for cyt c absorption (A550 - A540 nm) was taken to be 19.1 mM-1 cm-1. Recording the Absorbance Spectra of Mitochondria in the Presence of Acrebol and Inhibitors of Complex III. Absorbance spectra (450-700 nm, step 0.12 nm) of suspensions of rat liver mitochondria, in the presence of acrebol and/or of inhibitors of the respiratory complex III, were recorded in 2 mL plastic cuvettes using a double-beam recording spectrophotometer UV-3000 (Shimadzu, Japan). The suspension contained ∼2.5 mg of mitochondrial protein per mL. The second beam (520 nm), which passed the sample cuvette, was used as a reference for the correction of light scattering. The resulting spectra were averaged from four scans. Measurement of Superoxide Anion Production in the Mitochondria. The production of superoxide anion was measured using the selective chemiluminescent superoxide probe, 3,7-dihydro-2methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) (32). Chemiluminescence was detected with the Victor3 multiplate reader. Sperm Cell Toxicity Assessments. For testing, the compounds were dissolved in 1 µL of methanol and dispensed in 200 µL of extended boar semen, and after 5 min of incubation in a thermoblock at 37 °C, they were inspected for motility of the sperm. This was done using a phase-contrast microscope (with a heated stage) in triplicate as described by Andersson et al. (33). For calibration of the bioassay, valinomycin was used. Valinomycin (2 ng mL-1 of sperm suspension) completely inhibited sperm motility after 5
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Figure 1. Effects of acrebol on the transmembrane potential (∆Ψm) and the respiration of isolated rat liver mitochondria. (A) The respiration rate (Clark oxygen electrode) and ∆Ψm (TPP+ electrode) in mitochondria were simultaneously measured in standard KCl-based medium, which contained 5 mM glutamate, 5 mM malate, and 1 µM TPP+. Where indicated, RLM (1 mg protein mL-1), acrebol (125 and 350 ng mL-1), 5 mM succinate, 200 µM TMPD, 2 mM ascorbate, 1 mM NaCN, and 1.5 mM MgATP were added. (B) Inhibition of uncoupled respiration by acrebol. RLM was incubated for 2 min before the other additions in the medium containing 5 mM succinate, 1 µM rotenone, and 500 µM EGTA. The respiration rate was measured in mitochondria uncoupled by 500 nM FCCP and injected into the suspension 1 min before (squares, 9) or 5 min after (circles, b) acrebol (concentrations indicated on the horizontal axis). The steady-state respiration rate was recorded. The data of the curve are the means ( SEMs of three independent experiments (n g 3).
min of exposure. The integrity of the plasma membrane was probed by propidium iodide staining and potential changes (∆Ψ) by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining (1, 34, 35). JC-1 is a membranepenetrating dye that fluoresces orange in membranes with high ∆Ψ and fluoresces green when ∆Ψ is low. The ATP content in the sperm cells was measured using the ATP Biomass Kit HS of BioThema AB (Haninge, Sweden) following the manufacturer’s protocol. MIN-6 Cell Toxicity Assays. Mouse pancreatic β-cell line MIN-6 (35) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% (v/v) fetal bovine serum, 50 µM β-mercaptoethanol, penicillin (100 units mL-1), streptomycin (0.1 mg mL-1), 20 mM HEPES (pH 7.4), and 20 mM MgCl2 at 37 °C in a water-saturated atmosphere of 5% CO2 and 95% (v/v) air. The cells were seeded on culture slides (BioCoat Variety Pack, Erembodegem, Belgium) and incubated for 2 days before exposure to toxins. After the toxins were added, the slides were incubated under the same conditions. To assess the number of cells with intact plasma membranes, membrane blebbing, and fragmented nuclei, the exposed cells (no fixative used) in chambers containing 500 µL of the medium were stained with 0.7 µg Calcein-AM (1 mg mL-1 DMSO), 5 µg of propidium iodide (1.6 mg mL-1 of water), and 10 µg of Hoechst 33342 (10 mg mL-1 of water) (Molecular Probes, Invitrogen, Carlsbad, CA) (36, 37). After 20 min in the dark, free dyes were removed by washing with RPMI 1640 medium (Sigma), and the cells were allowed to equilibrate at room temperature for 10 min and then inspected with fluorescence microscope (excitation 450-490 nm, band-pass; 520 nm long pass for emission). The cells with intact plasma membrane and membrane blebbing fluoresced green, and the cells with damaged plasma membrane fluoresced red. Fragmented apoptotic nuclei were stained blue by Hoechst 33342, and necrotic nuclei were stained red (dead) by propidium iodide. Filters of 330-380 (excitation) and 480 nm (emission) were used. The release of cyt c from MIN-6 mitochondria in intact cells was assessed using SelectFX Alexa Fluor 488 Cytochrome c Apoptosis Detection Kit (Invitrogen) following the manufacturer’s manual. Statistical Procedure. The data shown represent the means ( standard errors of means (SEM) or are the means of at least three experiments. P values were derived by Student’s t test. Chemicals. Alamethicin, antimycin A, myxothiazol, rotenone, valinomycin, Trizma Base, HEPES, EGTA, BSA, carbonyl cyanidep-(trifluoromethoxy)phenyl-hydrazone (FCCP), TPP+, sucrose, mannitol, malate and glutamate, MCLA, and dithionite were
obtained from the Sigma-Aldrich Corp. (St. Louis, MO). The other chemicals were of analytical grade, purchased from the local suppliers.
Results Acrebol Inhibited the Respiratory Chain Complex III in Rat Liver Mitochondria without Affecting the Mitochondrial Permeability for Solutes. Acrebol, isolated from the indoor fungus A. exuViarum BMB4, was purified guided by its inhibitory action on the motility of boar spermatozoa. The purified substance, consisting of two closely similar peptides, inhibited sperm motility at a concentration of less than 200 nM (5). Its effects on isolated rat liver mitochondria were studied. Figure 1A shows that acrebol at a concentration of 475 ng mL-1 (125 + 350 ng mL-1, equivalent to ca. 280 nM) strongly inhibited the respiration of mitochondria on glutamate and malate (substrates that provide complex I with NADH) and caused the dissipation of the ∆Ψm. The addition of succinate, a substrate of complex II of the respiratory chain, restored neither the respiration nor the ∆Ψm. Ascorbate and N,N,N′,N′tetramethyl-p-phenylenediamine (TMPD), which transfer electrons to cyt c and cyt c oxidase, restored both the respiration and the ∆Ψm. A subsequent addition of cyanide (1 mM) caused an immediate dissipation of the transmembrane potential, ∆Ψm (TPP+ electrode). The respiration on TMPD and ascorbate was inhibited almost immediately thereafter: Only e80 nmol of oxygen (out of the ca. 250 nmol present) per mL was consumed (O2 electrode). The apparent delay of 1/2 min in Figure 1A in the cessation of oxygen consumption after cyanide had been added may have been due to the Clark electrode, which is known to be more inert than the TPP+ electrode. Figure 1B shows the dependence of the rate of uncoupled respiration on the concentration of acrebol in the incubation medium. Acrebol was added 5 min before or 2 min after the addition of the uncoupler of oxidative phosphorylation, FCCP. The concentration of acrebol that caused a 50% inhibition of the FCCP uncoupled respiration (O2 electrode) was 80 and 110 ng mL-1 per mg protein with (circles) and without (squares) preincubation, respectively. Complete inhibition was observed in the presence of about 350 ng of pure acrebol and 1 mg of mitochondrial protein mL-1. Thus, acrebol extremely effectively inhibited the respiratory
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Figure 2. Effect of acrebol on the volume of isolated rat liver mitochondria and the redox state of the mitochondrial pyridine nucleotides in media of different compositions. (A) Mitochondria (0.5 mg protein mL-1) were added just before the measurements in KCl- (KCl), NaCl- (NaCl), KNO3(KNO3), and sucrose-mannitol-based medium (SM) supplemented with respiratory substrates (5 mM glutamate and 5 mM malate) and 500 µM EGTA. Where indicated, acrebol (200 ng mL-1), valinomycin (Valino) (25 ng mL-1), or alamethicin (Alam) (2.5 µg mL-1) was added. Standard traces of one of at least three identical experiments are shown. (B) Mitochondria (0.5 mg protein mL-1) were added (just before the measurements) to KCl-based medium supplemented with 5 mM glutamate and 5 mM malate. Where indicated, acrebol (200 ng mL-1), 500 nM FCCP, valinomycin (Valino) (25 ng mL-1), alamethicin (Alam) (2.5 µg mL-1), or antimycin A (Ant A) (250 ng mL-1) was added. PN, pyridine nucleotides. The data shown in the traces are the means ( SEMs (n ) 4) of one experiment of three identical experiments.
chain at the level of ubiquinol cyt c reductase; that is, it most likely blocked the complex III of the mitochondrial respiratory chain. We compared the effects of acrebol to those of well-known complex III inhibitors, antimycin A and myxothiazol, and of the pore-forming peptaibol alamethicin on the volume of mitochondrial matrix and the redox state of mitochondrial pyridine nucleotides. Figure 2A shows that alamethicin induced high amplitude swelling of isolated rat liver mitochondria in sucrose-mannitol-based media. In contrast, acrebol induced no high amplitude swelling of mitochondria at concentrations (200 ng and 0.5 mg of mitochondrial protein mL-1) sufficient for the inhibition of mitochondrial respiration and the dissipation of ∆Ψm. The situation remained the same when the salt-free sucrose-mannitol-based medium was replaced by [K+][Cl-]-, [Na+][Cl-]-, and [K+][NO3-]-based media. In the presence of the K+ ionophore valinomycin, acrebol slightly decreased the rate of high amplitude swelling of rat liver mitochondria in K+-containing medium. Similar inhibition of valinomycin-dependent swelling was observed in the presence of the inhibitors of the complex III of the respiratory chain, antimycin A and myxothiazol, and of FCCP, the uncoupler of oxidative phosphorylation (not shown). Figure 2B shows that in K+containing medium, acrebol preserved the reduced state of the mitochondrial pyridine nucleotides, similarly to antimycin A but in contrast to alamethicin, FCCP, or valinomycin. The effect of acrebol in sucrose-mannitol-, NaCl-, and KNO3-based media was the same as in the K+ medium in Figure 2B. These data indicate that, at the concentrations used, acrebol did not form pores or ion channels for K+, Na+, or Cl-. Acrebol also did not transfer divalent cations (Ca2+ and Mg2+), as indicated by the Ca2+-selective electrode and spectrophotometry, respectively (not shown). Summarizing, these results show that the mitochondrial action of acrebol resembles that of antimycin A and myxothiazol, inhibitors of the respiratory chain complex III. We investigated the potential of acrebol to induce the release of cyt c from mitochondria by comparing it with those of known inhibitors of the respiratory chain (rotenone and antimycin A), ionophoric peptides (alamethicin and valinomycin), and with that of Ca2+, an opener of the mitochondrial permeability transition pore. The results in Figure 3 show that exposure to acrebol, to antimycin A, or to rotenone caused no cyt c release beyond the control level, whereas alamethicin, valinomycin, and
Figure 3. Release of cyt c from isolated rat liver mitochondria in the presence of pore-forming agents, inhibitors of the respiratory chain, and acrebol. RLM (12 mg per 6 mL) was incubated as described in the Experimental Procedures and, if indicated, in the presence of 1 mM EGTA (EGTA). After 1 min, CaCl2 (Ca2+, 200 µM), alamethicin (Alam, 60 µg mL-1), valinomycin (valino, 50 ng mL-1), antimycin A (AntA, 1 µg mL-1), rotenone (Rot, 1 µg mL-1), or acrebol (500 ng mL-1) was added. After 10 min of exposure, the mitochondria were collected, and the quantity of cyt c in the supernatants was analyzed. The data shown in the columns are the means ( SEMs of three independent experiments (n g 3). The significant difference (P < 0.05) between control and toxin-containing samples is indicated by an asterisk.
Ca2+ (i.e., the agents that induced mitochondrial high amplitude swelling, Figure 2A) liberated, as expected, considerable amounts of cyt c from the mitochondria. Therefore, the inhibition of the respiratory chain by acrebol could not be ascribed to the release of cyt c due to any mechanism and was most likely related to direct inhibition of the complex III. The absorption spectra of rat liver mitochondria suspensions in the presence of acrebol and known inhibitors of inner (Qi) and outer (Qo) quinone-binding centers of complex III of the respiratory chain are presented in Figure 4. As shown in the figure, acrebol caused the oxidation of the mitochondrial cyt c (the decrease of absorbance at 550 nm) and reduction of cytochrome b (the increase of absorbance at 560 nm), similarly to antimycin A and myxothiazol. Similar to myxothiazol, which inhibits the Qo-center, acrebol also caused the appearance of an absorption band with a maximum at 558 nm. Antimycin A, which blocks electron transport at the Qi-center, caused appearance of the band with a maximum at about 562 nm. Thus, one
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Figure 4. Standard differential absorption spectra of rat liver mitochondria suspensions in the presence of acrebol and the known inhibitors of complex III, antimycin A, and myxothiazol. (A) Differential spectra (reduced minus oxidized) of cytochromes in the presence of 5 mM succinate (Succinate) with myxothiazol (Myx, 500 ng mL-1), antimycin A (AntA, 500 ng mL-1), acrebol (Acrebol, 500 ng mL-1), or dithionite (Reduced). Spectra of the oxidized cytochromes were taken in the absence of substrates and in the presence of potassium ferricyanide. (B) Differential spectra of those presented in panel A (spectra in the presence of the inhibitor of complex III minus spectra during the steady-state oxidation of succinate). The spectra are averages of at least three identical experiments.
myxothiazol, which prevents ubisemiquinone formation in the Qo-center and hence the extrusion of the superoxide anion into the intermembrane space of mitochondria, acrebol markedly diminished the superoxide-dependent chemiluminescence of MCLA. The uncoupler of oxidative phosphorylation FCCP, which decreases the lifetime of ubisemiquinones in the respiratory chain, also inhibited the production of superoxide.
Figure 5. Production of superoxide anion by rat liver mitochondria in the presence of acrebol, antimycin A, and myxothiazol. Immediately before measurements, the mitochondria (0.3 mg protein mL-1) were placed in standard KCl-based medium supplemented with the respiratory substrates, 20 µM MCLA, and one of the following: acrebol (200 ng mL-1), myxothiazol (Myx, 100 ng mL-1), antimycin A (AntA, 500 ng mL-1), FCCP (500 nM), or none. Measurements were performed in 96 well plates at 30 °C. The traces shown are the means ( SEMs (n ) 4) of one experiment of three identical experiments. The traces are labeled as follows (from top to bottom); b (circles), antimycin A; 9 (squares), control; 2 (triangles up), acrebol; 1 (triangles down), myxothiazol; and ( (diamonds), FCCP.
may suggest that acrebol, similarly to myxothiazol, acts on the Qo-center of complex III but not on the Qi-center, as antimycin A does. Data obtained upon measurements of the level of superoxide anion with chemiluminescent probe MCLA, which is most efficient in the intermembrane space of mitochondria, were in line with this suggestion. Figure 5 shows that antimycin A, which stabilizes the autoxidizable ubisemiquinone in the Qocenter of complex III, strongly stimulated the production of superoxide anion in the outer compartments of mitochondria, as indicated by chemiluminescence of MCLA. Similar to
Suppression of sperm cell motility by acrebol is connected to the loss of mitochondrial ATP. We investigated if a short exposure to purified acrebol, at concentrations sufficient to inhibit the progressive motility in 100% of the sperm cells, affected the sperm cell ATP content. Figure 6 shows that exposure to 250 ng mL-1 of acrebol, as well as antimycin A (500 ng mL-1), FCCP (500 nM), or oligomycin (500 ng mL-1), stopped the progressive movement of boar spermatozoa. The inhibition was rapid and irreversible but was not, except for the presence of FCCP, accompanied by any dissipation of the ∆Ψm (indicated by JC-1 staining). The total ATP content of sperm cells, however, was reduced in the presence of acrebol, as well as antimycin A and FCCP, by ca. 50%, from 1.5 fmol per sperm cell to 0.7-0.8 and to 0.6-0.7 fmol per sperm cell, after 10 min and 2 h of incubation, respectively. Oligomycin inhibits the mitochondrial ATPase and thus prevents the hydrolysis of cytosolic ATP. When oligomycin was added to the sperm suspension after incubation with acrebol or antimycin A, it caused transient stimulation of the progressive movement of spermatozoa, which was accompanied by an irreversible decrease in ∆Ψm. The intracellular concentrations of ATP in the presence of acrebol plus oligomycin and antimycin A plus oligomycin were higher than in the presence of the complex III inhibitors alone. These data clearly indicate that the toxic effect of acrebol at low concentrations (∼100-250 ng mL-1) on the sperm cells was related to the inhibition of the respiratory chain and the depletion of cellular ATP. The latter is due to the reversion of the activity of mitochondrial ATPase and the continuing hydrolysis of ATP by the dynein ATPase.
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Figure 6. Effect of acrebol on the progressive motility, ATP content, and the mitochondrial transmembrane potentials of boar sperm cells. Partial recovery by F0F1-ATPase inhibitor. The progressive movement, transmembrane potential (JC-1 staining), and intracellular ATP were assessed as described in the Experimental Procedures. The final concentrations of toxins in the semen were as follows: acrebol, 250 ng mL-1; oligomycin (Oligo), 500 ng mL-1; antimycin A (AntA), 500 ng mL-1; and FCCP, 500 nM. In the presence of acrebol (Acrebol + Oligo) and antimycin A (Ant A + Oligo), oligomycin was added to the semen just before the microscopic examination. Asterisks at plus signs indicate that the stimulation of motility was short-term. The data shown in the columns are the means ( SEMs of three independent experiments done in three parallel experiments. The significant difference (P < 0.05) between control and toxin-containing samples is indicated by a doubled asterisk. Numbers under the X-axis indicate the exposure time of the toxins.
Acrebol Killed Mouse Insulinoma MIN-6 Cells in a Mode Similar to That by Myxothiazol and by Antimycin A. The sperm cells lack the ability to proliferate. Therefore, mouse insulinoma MIN-6 cells were used as a model to explore the impact of acrebol on the viability of cells that are highly dependent on mitochondrial ATP. As shown in Table 1, MIN6 cells were highly susceptible to the agents that disrupt mitochondrial functions. Exposure to acrebol (250 ng and 4.5 µg), antimycin A (500 ng), myxothiazol (100 ng), oligomycin (500 ng), valinomycin (25 ng), or alamethicin (500 ng) per mL initiated fast cell death as indicated by propidium iodide staining, observable already after 4 h of incubation. After 24 h of incubation in the presence of the tested substances (excepting acrebol at low concentration), all cells were stained red, indicating 100% death. Cells that survived 24 h of incubation with a low concentration (250 ng mL-1) of acrebol subsequently (48 h) recovered to normal viability and rates of growth. Valinomycin and alamethicin, but not acrebol or the other inhibitors, induced a massive release of cyt c from the mitochondria of MIN-6 cells that is prerequisite for apoptosis. However, the cell death initiated by all agents developed without nuclear fragmentation (Hoechst 33342 staining), blebbing, or shrinkage of the cytosol, indicating that the mechanism was necrosis.
Discussion The unique toxic actions of acrebol, the heat-stable and lipophilic peptaibol from A. exuViarum BMB4 (5), on mammalian cells are reported in this paper. We report that acrebol displays a unique toxic effect on mammalian cells and on mitochondria; it is the first inhibitor of the mitochondrial respiratory chain among the known peptaibols. The toxic effect of acrebol was connected to the inhibition of the respiratory chain at the level of complex III (see Figure 1),
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without changes in ionic and solute permeability of the mitochondrial membranes (see Figure 2). This was indicated by the arrest of uncoupled respiration, the absence of mitochondrial swelling in various media, and the absence of the release of cyt c in media with high ionic strength (see Figure 3). Inhibition of the mitochondrial respiratory chain has been reported for other peptaibols, for example, alamethicin (19, 38, 39), but that inhibition was shown to be a consequence of high amplitude swelling and the release of cyt c from the intermembrane space (19, 38). Adding exogenous cyt c restored the respiration, indicating that the effect of peptaibols on the respiratory chain was indirect (19, 39). In contrast, the inhibition by acrebol of complex III was direct and potent with an IC50 ∼ 80 and 110 ng (50 and 70 nM) with and without preincubation, respectively, and ca. 350 ng caused a complete halt (see Figure 2B). Taking into account the estimated content of complex III in rat liver mitochondria, 50-80 pmol per mg of protein (40), acrebol inhibited complex III with an apparent stoichiometry of 1-2 mol of acrebol per mol of complex III. Thus, acrebol is almost as potent of a complex III inhibitor as antimycin A and myxothiazol (41, 42). Analysis of the absorption spectra of rat liver mitochondria suspensions in the presence of acrebol and other inhibitors of respiratory complex III demonstrated that acrebol, similar to myxothiazol, specifically inhibited the Qo-center of the complex (41, 42), in contrast to antimycin A, which inhibits the Qi-center (see Figure 4) (43). Indirect evidence for the myxothiazol-like inhibition of complex III by acrebol came from the study of the rates of superoxide anion production in the outer compartments of mitochondria. It has long been known that antimycin A, which blocks the Qi-center of complex III (44), strongly activates the superoxide anion production in the Qo-center (45-47). The effect is due to the prevention of ubisemiquinone oxidation by FeSRieske and cyt c1 and, thus, switching over to the oxidation by molecular oxygen (45, 46). Myxothiazol, which is known to prevent the first one-electron oxidation of ubiquinol in the Qo-center (48), precludes the formation of ubisemiquinone and thus its autoxidation and superoxide generation (46, 49). There is evidence indicating that myxothiazol still can activate superoxide anion formation but only in the Qi-center (50-52). However, superoxide anion is extruded to the matrix side of the membrane and is not detectable by probes that cannot easily penetrate the mitochondrial membranes and compete with manganese-containing superoxide dismutase (MnSOD) in the matrix, such as the MCLA used in this study (52, 53). In our studies, acrebol, similar to myxothiazol and the uncoupler FCCP, which also decreases the lifetime of ubisemiquinone (53, 54) but in contrast to antimycin A, decreased the level of superoxide anion in the intermembrane space (see Figure 5). The data in the present study indicate that acrebol is a unique toxin in the family of peptaibols. In contrast to the majority of complex III inhibitors from various fungi and bacteria, acrebol is not a quinone-like compound or a macrolide but an amphipatic peptide (shown in Figure 7). The known peptaibols (55) that are closely similar to acrebol are immunosuppressive and neuroleptic cephaibols P and Q from Acremonium tubakii, acetyl-Phe-Iva/Val-Gln-Aib-Ile-Thr-Aib-Leu-Aib-Hyp-Gln-AibHyp-Aib-Pro-Phe-SerOH, and acetyl-Phe-Iva-Gln-Aib-Ile-ThrAib-Leu-Aib-Pro-Gln-Aib-Hyp-Aib-Pro-Phe-SerOH, respectively (56, 57). As these peptaibols and the majority of other members of peptaibol subfamily 3 (11, 55), acrebol [acetylPhe-Iva/Val-Gln-Aib-Ile-Thr-Leu-Aib/Val-Pro-Aib-Gln-ProAib-(X-X-X)-SerOH] has conserved threonine in position 6 and
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Table 1. Signs of the Death of MIN-6 Cells Induced by Acrebol and Other Microbial Toxins damage observed (% of the cells ( SEMc) exposure time
inhibitor
4 h
unexposed vehicle onlya antimycin A myxothiazol acrebol acrebol valinomycin alamethicin unexposed vehicle onlyc antimycin A myxothiazol acrebol acrebol valinomycin alamethicin unexposed vehicle onlyc acrebol
24h
48h
ng/mLb
500 100 250 2350 25 2500 500 100 250 2350 25 2500 250
death
fragmented nuclei
blebs on the plasma membrane
delocalized cyt c
6.5 ( 2.5 7.0 ( 3.0 11.4 ( 3.9 13.4 ( 3.5 6.3 ( 4.5 10.4 ( 2.1 15.6 ( 5.1 12.8 ( 3.3 7.8 ( 1.5 11.3 ( 2.6 100 100 54.6 ( 9.0 94.7 ( 0.5 100 100 6.3 ( 2.3 7.5 ( 2.5 19.9 ( 8.4
1.74 ( 0.45 0.25 ( 0.25 1.25 ( 0.25 1.75 ( 0.25 1.04 ( 0.95 2.0 ( 0.6 2.5 ( 0.65 2.0 ( 1.1 3.0 ( 0.7 2.75 ( 0.6 2.25 ( 1.11 2.0 ( 1.2 3.6 ( 0.7 2.33 ( 0.4 4.5 ( 0.5 5.0 ( 0.7 2.33 ( 0.33 2.67 ( 0.88 3.33 ( 0.33
0.67 ( 0.33 0.75 ( 0.25 1.25 ( 0.48 1.0 ( 0.4 3.1 ( 1.3 1.0 ( 0.0 3.5 ( 0.65 1.5 ( 0.3 0.5 ( 0.3 0.75 ( 0.25 0.25 ( 0.25 1.0 ( 0.25 0.6 ( 0.6 1.25 ( 0.25 1.38 ( 0.41 2.0 ( 1.25 1.25 ( 0.6 1.5 ( 0.3 1.5 ( 0.9
3.0 ( 1.1 1.0 ( 0.41 1.5 ( 0.3 2.0 ( 1.1 3.5 ( 0.6 4.5 ( 0.96 11.5 ( 4.3 18.6 ( 7.8 4.5 ( 2.5 3.5 ( 2.67 2.7 ( 1.5 1.75 ( 0.63 4.0 ( 1.7 7.5 ( 2.67 90.6 ( 5.5 95.3 ( 2.5 4.67 ( 0.33 5.0 ( 1.5 6.0 ( 2.0
Ethanol. The concentrations of test substances at the time of addition. c The values are the means ( SEMs of 2-3 independent experiments, in which three areas with g100 cells each were examined (n g 6). a
b
Figure 7. Structures of decylubiquinone, inhibitors of complex III of the respiratory chain, and the partial structure of acrebol A. The figure shows residues 1-13 of one of the closely similar two peptaibols. Residues 14-16 are unknown, and the C-terminal residue is serine. This figure is based on data described by Andersson et al. (5).
glutamines in the positions 3 and 11 (5). On the other hand, imino acid residues of proline are in positions 9 and 12 (5), instead of 10 and 13, as is usual for peptaibols from this group (11). It is concluded that the mechanism of toxic effects of acrebol on the mammalian cells (boar spermatozoa, mouse insulinoma) involves inhibition of the complex III of the mitochondrial respiratory chain. The subsequent depletion of cellular ATP was due to normal metabolic processes (including hydrolysis of ATP by the dynein ATPase in sperm cells) and reversion of ATP synthesis by mitochondrial F1F0-ATPase, resulting in immobilization of spermatozoa and necrosis-like death of the MIN-6 cells (see Figure 6 and Table 1). No sign was found of mechanisms other than the inhibition of complex III to explain
the toxicity of acrebol toward the mammalian cells. The fact that oligomycin alone inhibited, whereas added after acrebol it transiently stimulated, the boar sperm cell motility (Figure 6) may be explained by the plasticity of glycolysis. Because the activity of dynein ATPase in boar spermatozoa is strongly dependent on ATP produced via oxidative phosphorylation in mitochondria, inhibition of F1F0-ATPase by oligomycin stopped the cell motility. Inhibition of respiratory chain by acrebol or antimycin initiated the hydrolysis of glycolytic ATP by F1F0ATPase that, in turn, should stimulate the glycolysis and further inhibit dynein ATPase activity via ADP. Oligomycin, added after acrebol or antimycin, immediately stopped the ATP hydrolysis by mitochondrial F1F0-ATPase, while the inhibition of ATP production in glycolysis was delayed. This transient
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increase in ATP concentration is, presumably, enough for brief stimulation of sperm cell motility. It is noteworthy that simultaneous exposure of MIN-6 cells to agents that liberated apoptogenic cyt c from the mitochondria (alamethicin and valinomycin) and to agents that preserved intactness of the mitochondrial membranes (acrebol, antimycin A, and rotenone) caused the same type of cell death, necrotic cell death. This may be explained by the competition of apoptotic and insulin-producing/extruding pathways for the cellular ATP (25, 58, 59). When mouse insulinoma cell line MIN-6 was exposed to a low concentration of acrebol, the proliferation of MIN-6 cells was only transiently arrested. Complex III is in relative excess in the respiratory chain in comparison with complexes I and II (60). The transient arrest may result from a stoichiometric and tight binding of acrebol to its target in complex III, allowing the unbound complex III to support the onset of proliferation of the insulinoma cells upon extended exposure (Table 1). Taking into account that acrebol-producing fungi and their metabolites may be found in places of human life and activity, there is a possibility of acute and chronic intoxication by acrebol. Acute intoxication may especially damage the tissues in which oxidative phosphorylation makes the greatest contribution to total ATP synthesis. For chronic intoxication several scenarios are possible as follows: (1) suppression of fatty acid oxidation in the mitochondria, vital, for example, for the heart muscle; (2) suppressed oxidation of free fatty acids, leading to an increase of their levels and uptake and increased synthesis of triglycerides and diacyl glycerol (DAG), the main activator of the PKCs (protein kinase C) (61, a review). Free fatty acids synergize with DAG in the activation of the classical PKCs as well as the PKC-θ responsible for mediating the regulation of many functions, for example, the insulin receptors (62, 63) and human T-cell-mediated immune responses (for reviews, see refs 64 and 65); (3) damage to endocrine cells that are highly dependent on mitochondrial activity (25, 58, 59, 66, 67); and (4) promotion of undesirable tissues or cells adapting to insufficient oxidative phosphorylation by upregulating glycolysis, for example, malignant cells and pathogenic microorganisms. Acknowledgment. The project was supported by the Academy of Finland Photobiomics Grant (118637). L.K. is the grantee of the Ja´nos Bolyai Research Scholarship (Hungarian Academy of Sciences). We thank Prof. Mikhail Verkhovsky from the Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, for the expert advice. We thank the Viikki Science Library and the Faculty Instrument Centre for the expert services and Leena Steininger, Hannele Tukiainen, and Tuula Suortti for many kinds of help.
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(6) (7) (8) (9) (10)
(11) (12)
(13)
(14) (15) (16) (17) (18)
(19)
(20)
(21) (22)
(23) (24)
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