Daphnetoxin Interacts with Mitochondrial Oxidative Phosphorylation and Induces Membrane Permeability Transition in Rat Liver

• Abstract
• Introduction
• Materials and Methods
• Plant material
• Isolation and identification of daphnetoxin
• Isolation of hepatocytes and rat liver mitochondria
• Mitochondrial respiratory activity
• The mitochondrial transmembrane potential
• ATPase activity
• Quantification of adenine nucleotides
• Mitochondrial swelling
• Chemicals
• Treatment of the data.
• Results
• Discussion
• Acknowledgements
• References

Abstract

The effects of daphnetoxin on isolated rat liver mitochondria and freshly isolated rat hepatocytes were investigated. Daphnetoxin (in the μM range) increased mitochondrial state 4 respiration and decreased both state 3 and FCCP-uncoupled respiration. The transmembrane potential was strongly depressed by daphnetoxin in a concentration-dependent manner. The protonophoric activity of daphnetoxin was evidenced by the induction of mitochondrial swelling in hyposmotic K⁺ acetate medium in the presence of valinomycin. In isolated hepatocytes, daphnetoxin decreases intracellular ATP and simultaneously increases ADP and AMP concentrations. The addition of uncoupling concentrations of daphnetoxin to Ca²⁺-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Mitochondrial swelling in the presence of Ca²⁺ was prevented by cyclosporine A and was drastically inhibited by catalase and dithiothreitol, indicating the participation of mitochondrial generated reactive oxygen species in this process. From this study we can conclude that the bioenergetic lesion promoted by daphnetoxin seems to be sufficient to explain the lethal hapatocyte injury.

Introduction

Plants from the family Thymelaeaceae are known to cause a wide range of biological effects, namely toxic, irritant, carcinogenic, cocarcinogenic and antineoplastic [1]. As some species are used in folk medicine in tropical Africa and in China, many compounds have already been isolated and chemically characterized [2]. The systematic fractionation of extracts from Daphne gnidium L. has led to the isolation of daphnetoxin. A comparative study of two similar molecules, the natural products daphnetoxin and mezerein, has shown that daphnetoxin is also an activator of classical protein kinase C isoforms, but differs from mezerein in its selectivity [3]. This difference has been suggested as a possible reason for the antileukemic properties observed with mezerein [4], but not with daphnetoxin.

At present, data concerning the effects of daphnetoxin on mitochondrial bioenergetics have not been reported. Interference with mitochondrial bioenergetics is known to be part of the process of cell injury by assorted agents and by a variety of mechanisms [5]. Mitochondria support the energy-dependent regulation of many cell functions, namely intermediary metabolism, protein folding, ion regulation, cell motility and cell proliferation [6]. Mitochondrial dysfunction can lead to apoptotic cell death and to some neuronal degenerative diseases [7]. Furthermore, the mitochondrion is a good model for studying the cell toxicity of many xenobiotics, since data obtained from such studies are generally well correlated with cytotoxicity parameters reported in cell cultures and whole organisms [8]. In this paper we study the effect of daphnetoxin, a natural diterpene isolated from Daphne gnidium L., on rat liver mitochondrial bioenergetics. We demonstrate the protonophoric properties of daphnetoxin and its role in inducing non-specific inner mitochondrial membrane permeabilization in the presence of Ca²⁺.

Materials and Methods

Plant material

The stem bark of Daphene gnidium L. was collected in March 2003 in the region of Régua, Portugal. A voucher specimen (No. 10 353) has been deposited at the Herbarium of the University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.

Isolation and identification of daphnetoxin

The fresh stem bark of D. gnidium (1.5 kg) was extracted with chloroform at room temperature for 15 days. The chloroform extract was concentrated under vacuum and the resultant solid (15.7 g, 1.0 %) was then extracted with 0.5 L of n-hexane. The n-hexane insoluble fraction (11.2 g, 0.75 %) was subjected to column chromatography (CC) using silica gel 60 (230 - 400 mesh; 300 g) with EtOAc/n-hexane (1 : 9, 2 : 8, 3 : 7, and 4 : 6) providing 70 fractions (150 mL each). Analysis by TLC (silica gel 60 F₂₅₄, 0,2 mm thick) using daphnetoxin as standard was performed. Fractions containing daphnetoxin (65 - 67) were combined, concentrated under vacuum and subjected to preparative thin layer chromatography (PTLC, silica gel 60, 2 mm thick) with EtOAc/n-hexane (1 : 1) to give 138 mg of pure daphnetoxin (whitish crystals). Daphnetoxin was identified by IR, NMR (¹H and ¹³C) and mass spectra, melting point and specific optical rotation ([α]_D ²⁵: + 35°; c 0,06, CHCl₃). All data were in accord with those described in the literature [9].

Isolation of hepatocytes and rat liver mitochondria

Adult Wistar rats (200 - 300 g) were used as the source of hepatocytes. The cells were isolated by collagenase perfusion of liver as described by Moldéus et al. [10]. The hepatocytes viability was more than 85 % as estimated by measurement of cytosolic lactate dehydrogenase (LDH) leakage from cells into the medium. Hepatocytes (10⁶ cells/mL) were suspended in Krebs-Henseleit buffer (pH 7.4). All incubations were performed at 37 °C under an atmosphere of 95 % O₂ and 5 % CO₂. Reactions were started by the addition of daphnetoxin (30 μM) and aliquots of incubation mixture were withdrawn at time intervals for the analyses of cellular biochemical parameters. The isolation was performed by conventional methods [11], with minor modifications. The homogenisation medium contained 0.25 M sucrose, 5 mM Hepes (pH 7.4), 0.2 mM EGTA [ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid] and 0.1 % fatty acid-free BSA. EDTA (ethylenediaminetetraacetic acid) EGTA and BSA were omitted from the final washing medium, which was adjusted to pH 7.2. The final concentration of the mitochondrial protein was determined by the biuret method [12] using BSA as standard. The study was conducted in accordance with the Animals Scientific Procedures Act (1986), with the approval of the local ethics committee and under project license number PPL60/2362.

Mitochondrial respiratory activity

The oxygen consumption of isolated mitochondria was measured polarographically using a Clark-type oxygen electrode connected to a suitable recorder in a closed water-jacketed 1.0 mL chamber with magnetic stirring, at 25 °C. The standard respiratory medium consisted of 130 mM sucrose, 50 mM KCl, 5 mM MgCl₂, 5 mM KH₂PO₄, and 5 mM Hepes, pH 7.2. Daphnetoxin was added in aliquots (a few microlitres), from a concentrated ethanolic solution (1 M, pH 7.0), to 1 mL of the standard respiratory medium supplemented with mitochondria (0.8 mg protein) and allowed to incubate for 5 min before the addition of a respiratory substrate, i. e., before the beginning of the respiratory activity. State 4 was elicited after a phosphorylation of 50 μM, state 3 by adding adenosine 5′-diphosphate (ADP; 1 mM), and uncoupled respiration by adding 1 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP).

The mitochondrial transmembrane potential

The reactions were conducted under a continuous stream of O₂ to avoid anaerobisis. The transmembrane potential (Δψ) was estimated with a TPP⁺ electrode according to the equation of Kamo et al. [13], without correction for the ”passive” binding contribution of TPP⁺ to the mitochondrial membranes (as the purpose of the experiment was to show relative changes in the potential rather than absolute values). A matrix volume of 1.1 μL/mg mitochondrial protein was considered and valinomycin was used to calibrate the basal line. Reactions were carried out at 25 °C in 1 mL of the standard respiratory medium (the same medium as described for the oxygen consumption experiments) supplemented with 3 μM TPP⁺ and 0.8 mitochondrial protein. Calibration runs in the presence of daphnetoxin excluded any direct interference in the electrode signal.

ATPase activity

ATPase activity was determined by monitoring the pH change in association with ATP hydrolysis. The reaction was carried out at 25 °C, in 2 mL reaction medium (130 mM sucrose, 50 mM KCl, 5 mM MgCl₂, 0.5 mM HEPES, 2 μM rotenone, pH 7.2), freeze thawed mitochondria (0.5 mg) were added and incubated for 3 min with Triton X-100 (0.025 % v/v). The reaction was initiated by the addition of 2 mM Mg-ATP. The addition of oligomycin (1 μg/mg protein) to the medium completely halted the production of protons.

Quantification of adenine nucleotides

Adenine nucleotides were extracted using an alkaline extraction procedure and were separated by reverse-phase high performance liquid chromatography. The chromatographic apparatus was a Beckman-System Gold, consisting of a 126 model binary pump and a photodiode array detector, controlled by computer. The detection wavelength was 254 nm, and the column was Lichrospher 100RP-18 (5 mm) from Merck (Darmstadt, Germany). An isocratic elution with 100 mM phosphate buffer (KH₂PO₄), pH 6.5 and methanol 1 % was performed with a flow rate of 1 mL/min. The time required for each analysis was 5 min.

Mitochondrial swelling

Mitochondrial osmotic swelling was monitored by detecting turbidity, at 520 nm, on a Spectronic Genesys 2PC spectrophotometer, in a thermostatic chamber with magnetic stirring at 25 °C.

Chemicals

All reagents were of analytical grade for research. Daphnetoxin was isolated, in our laboratory, according to the description given above.

Treatment of the data.

Results are presented with the values of ± SD from at least three independent experiments. Statistical analyses were performed using two-tailed unpaired t-tests. A P value < 0.05 was considered statistically significant.

Results

The effect of daphnetoxin (Fig. [1]) on rat liver respiratory rates of state 4, state 3 and FCCP-stimulated (uncoupled) respiration were studied, in the presence of succinate as a respiratory substrate (Fig. [2]). A 5-min treatment of the mitochondrial suspension with 600 μM daphnetoxin results in the release of state 4 respiration with up to a 300 % increase in the rate of O₂ consumption and a decrease of about 30 % was observed in the rate of ADP-induced state 3 respiration. When the mitochondria were incubated in a reaction medium containing the uncoupler FCCP, succinate sustained oxygen uptake did not increase, only an inhibitory effect of about 50 % of the control was observed.

The addition of daphnetoxin to the succinate energised mitochondrial suspension depresses the membrane potential, as a function of toxin concentration (Fig. [3]). Due to the results obtained in state 3 respiration and in the depolarisation/repolarisation of the Δψ we decided to determine if the effect of daphnetoxin in state 3 respiration was exclusively due to a protonophoric effect or if daphnetoxin also interacts with the phosphorylative system. With this purpose in mind we studied the effect of daphnetoxin on mitochondrial ATPase activity (Fig. [4]), and the results obtained indicate that daphnetoxin, at all tested concentrations, carries out an inhibition on the ATPase activity. Changes in adenine nucleotides, reflecting energy metabolism in hepatic tissue, during exposure to daphnetoxin (30 μM) are shown in Table [1]. The value of cellular ATP decreases from 11.2 in the control to 6.2 nmol/10⁶ cells after 120 min of incubation. The decrease in ATP was reflected by a simultaneous increase in intracellular ADP and AMP; therefore, a dramatic impairment in cellular energetic processes is a predicted consequence. Addition of fructose, to force the glycolytic pathway to produce ATP delays the energy charge decrease. In order to prove the possible protonophoric properties of daphnetoxin, we performed mitochondrial swelling experiments in iso-osmolar medium. The K⁺ ionophore valinomycin was added in order to permit the movement of K⁺ across the mitochondrial membrane. Under these experimental conditions, mitochondrial swelling was observed in the presence of daphnetoxin in a dose-dependent manner (Fig. [5]). It has been demonstrated that FCCP-uncoupling of Ca²⁺-loaded mitochondria treated with Ruthenium Red (RuRed) leads to non-specific membrane permeabilisation, referred to as mitochondrial permeability transition pore (MPTP), specifically inhibited by cyclosporine A (CsA) [14]. Fig. [6] shows that, similarly to FCCP, daphnetoxin induces non-specific mitochondrial membrane permeabilisation, monitored as mitochondrial swelling in an isotonic sucrose based medium, in a dose-dependent manner. Catalase (2 μM) and dithiothreitol (DTT) (2 mM) drastically decreased the induced swelling by daphnetoxin in these conditions. Catalase, inactivated by boiling for 10 min, or the same protein concentration of BSA, did not cause inhibition of mitochondrial swelling (data not shown).

Discussion

Compounds isolated from Daphne species are known to exhibit toxic effects, daphnetoxin being one of the main toxic substances extracted from this species [9]. Nevertheless, despite the toxicity observed in many Daphne species, important anti-tumour activity has also been demonstrated, although these effects were not only observed in diterpenic [15] but also in coumarinic compounds [16]. Compounds that interact with mitochondrial membranes disrupting the coupling efficiency between oxidation and phosphorylation promote large bioenergetic deficits leading to the loss of several functions vital to the survival of the cell and the organism. Therefore, evaluation of disturbances induced by daphnetoxin on the mitochondrial function is important for understanding the molecular mechanisms of its toxicity.

The present study demonstrates that daphnetoxin strongly interacts with mitochondrial bioenergetics. Alterations of basic mitochondrial functions were monitored by the detection of changes induced in mitochondrial respiration and membrane energisation (Δψ). The enhancing effect of daphnetoxin in state 4 respiration accompanied with an inhibition on state 3 and in uncoupled respiration (Fig. [2]), together with the results obtained with Δψ (Fig. [3]) demonstrate that daphnetoxin acts as a typical mitochondrial uncoupler. Furthermore, the strongly observed swelling (Fig. [5]) also corroborates this idea. Oxygen consumption in the absence or presence of daphnetoxin, was completely inhibited by the addition of antimycin A (not shown), indicating that the oxygen consumption was exclusively from the respiratory activity. From the results obtained in the F₀-F₁ ATPase activity (Fig. [4]) it can be concluded that the observed effect on state 3 respiration was not exclusively due to the protonophore action promoted by daphnetoxin but also due to a specific effect on this enzyme.

The intracellular ATP level is decreased when hepatocytes are treated with daphnetoxin (30 μM), supporting the conclusion that daphnetoxin toxicity results essentially from a direct impairment of cellular bioenergetics, since addition of fructose to force the glycolytic pathway to produce ATP delays the cell energetic charge decrease.

The MPTP is a proteinaceous channel, formed when mitochondria are overloaded with calcium or in oxidative stress conditions [17]. It is characterised by mitochondrial membrane depolarisation, mitochondrial calcium release and an increase in non-specific permeability of the inner membrane to low molecular-weight solutes, leading to mitochondrial swelling and inhibition of oxidative phosphorylation. Induction of the permeability transition is widely implicated in the mechanisms by which many chemical compounds interfere with mitochondria bioenergetics and cell survival both in vitro [18] and in vivo [19]. A number of critical mechanisms have recently shown that mitochondria are involved in cell death and it has become increasingly clear that mitochondria play a central role in the processes that lead to cell death [20].

Fig. [6] shows that daphnetoxin induces non-specific mitochondrial membrane permeabilisation, monitored as mitochondrial swelling in isotonic sucrose-base medium, in a dose-dependent manner (lines c - g). This permeabilisation is inhibited by CsA (line a), catalase (dotted line) or DTT (line h), indicating that reactive oxygen species (ROS) generated by the mitochondria participate in the process. The MPTP opening induced by daphnetoxin was also confirmed by measuring extramitochondrial movements with a fluorescent calcium-sensitive probe Calcium Green 5-N (data not shown). A major target of mitochondrial [Ca²⁺] regulation, the permeability transition pore (MPTP), has been shown to be involved in the process of releasing apoptotic factors [21]. The opening of MPTP has been implicated in the release of cytochrome c and an apoptosis-inducing factor from mitochondria [22]. Therefore, on the basis of the MPTP induction by daphnetoxin, we intend to evaluate its cytotoxicity in cancer cell lines.

The results presented here lead to the conclusion that daphnetoxin acts as a classical uncoupler of oxidative phosphorylation and induces non-specific inner mitochondrial membrane permeabilisation of Ca²⁺-loaded mitochondria. These properties may help to explain some reports of animal deaths [23]. Furthermore, a large part of the toxicity attributed to the daphnetoxin could be the result of an effect on mitochondria in which reduced cellular homeostatic control causes cells to become non-viable.

Acknowledgements

This work was supported by the CECAV of University of Trás-os-Montes and Alto Douro, Portugal.

References

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• 8 Knobeloch L M, Blondin G A, Harkin J M. Use of submitochondrial particles for prediction of chemical toxicity in man.  Bull Environ Contam Toxicol. 1990;  44 661-8
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• 9 Stout G H, Balkenhol W G, Poling M, Hickernell G L. The isolation and structure of daphnetoxin, the poisonous principle of Daphne species.  J Am Chem Soc. 1970;  92 1070-1
  CrossrefPubMedSearch in Google Scholar
• 10 Moldéus P, Hogberg J, Orrenius S. Isolation and use of liver cells.  Methods Enzymol. 1978;  52 60-1
  PubMedSearch in Google Scholar

Dr. Francisco Peixoto

Department of Chemistry

University of Trás-os-Montes and Alto Douro

5000-911 Vila Real

Portugal

Fax: +351-25-935-0480

Email: fpeixoto@utad.pt

References

• 1 Felhauer M, Hecker E. Screening of Thymelaeaceae species for irritant, cocarcinogenic and antineoplastic activity.  Planta Med. 1986;  52 553-4
  PubMedSearch in Google Scholar
• 2 Kreher B, Neszmélyi A, Wagner H. Triumbellin, a tricoumarin rhamnopyranoside from Daphne mezereum .  Phytochemistry. 1990;  29 3633-7
  CrossrefPubMedSearch in Google Scholar
• 3 Saraiva L, Fresco P, Pinto E, Portugal H, Goncalves J. Differential activation by daphnetoxin and mezerein of PKC-isotypes alpha, beta 1, delta and zeta.  Planta Med. 2001;  67 787-90
  Thieme ConnectPubMedSearch in Google Scholar
• 4 Kupchan S M, Baxter R L. Mezerein: antileukemic principle isolated from Daphne mezereum L.  Science. 1975;  187 652-63
  PubMedSearch in Google Scholar
• 5 Wallace K B, Eells J T, Madeira V M, Cortopassi G, Jones D P. Mitochondria-mediated cell injury. Symposium overview.  Fundam Appl Toxicol. 1997;  38 23-37
  CrossrefPubMedSearch in Google Scholar
• 6 Wallace D C. Mitochondrial diseases in man and mouse.  Science. 1999;  283 1482-8
  CrossrefPubMedSearch in Google Scholar
• 7 Eckert A, Keil U, Marques C A, Bonert B, Frey C, Schüssel K, Müller W E. Mitochondrial dysfunction, apoptotic cell death, and Alzheimer's disease.  Biochem Pharmacol. 2003;  66 1627-34
  CrossrefPubMedSearch in Google Scholar
• 8 Knobeloch L M, Blondin G A, Harkin J M. Use of submitochondrial particles for prediction of chemical toxicity in man.  Bull Environ Contam Toxicol. 1990;  44 661-8
  CrossrefPubMedSearch in Google Scholar
• 9 Stout G H, Balkenhol W G, Poling M, Hickernell G L. The isolation and structure of daphnetoxin, the poisonous principle of Daphne species.  J Am Chem Soc. 1970;  92 1070-1
  CrossrefPubMedSearch in Google Scholar
• 10 Moldéus P, Hogberg J, Orrenius S. Isolation and use of liver cells.  Methods Enzymol. 1978;  52 60-1
  PubMedSearch in Google Scholar

Dr. Francisco Peixoto

Department of Chemistry

University of Trás-os-Montes and Alto Douro

5000-911 Vila Real

Portugal

Fax: +351-25-935-0480

Email: fpeixoto@utad.pt