Carbenoxolone Induces Oxidative Stress in Liver Mitochondria, Which Is Responsible for Transition Pore Opening
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内分泌学杂志 2005年第5期
Dipartimento di Chimica Biologica (M.S., V.B., A.T.), Università di Padova, Istituto di Neuroscienze del Consiglio Nazionale delle Ricerche, Unità per lo Studio delle Biomembrane, 35121 Padova; Dipartimento di Scienze Mediche e Chirurgiche-Endocrinologia (C.F., D.A.), Università di Padova, 35129 Padova; and Servizio di Endocrinologia (M.P.), Università di Sassari, 07100 Sassari, Italy
Address all correspondence and requests for reprints to: Professor Antonio Toninello, Dipartimento di Chimica Biologica, Università di Padova, Istituto di Neuroscienze del Consiglio Nazionale delle Ricerche, Unità per lo Studio delle Biomembrane, viale G. Colombo 3, 35121 Padova, Italy. E-mail: antonio.toninello@unipd.it.
Abstract
Carbenoxolone (Cbx), a derivative of glycyrrhetinic acid, which has been found to affect mineralocorticoid and glucocorticoid receptors, induces swelling and membrane potential collapse when added to Ca2+-loaded liver mitochondria at 10 μM concentrations.
These effects are strictly correlated with hydrogen peroxide generation, increase in oxygen uptake, and sulfhydryl and pyridine nucleotide oxidation. Cyclosporin A, bongkrekic acid, and N-ethylmaleimide completely abolish all the above-described effects, suggesting that Cbx can be considered an inducer of mitochondrial permeability transition by means of oxidative stress. Cbx can also trigger the apoptotic pathway because the above events are also correlated with the loss of cytochrome c. These effects are probably related to the conjugated carbonyl oxygen in C-11, which produces reactive oxygen species by interacting with the mitochondrial respiratory chain, mainly at the level of complex I but, most likely, also with complex III. The oxidative stress induced by Cbx, which is responsible for pore opening, excludes that this is related to a genomic effect of the compound.
Introduction
CARBENOXOLONE (CBX) IS the 3-hemisuccinate of glycyrrhetinic acid (GE), the active principle of licorice (for structure see Fig. 1); it has been used for 40 yr in the treatment of gastritis and ulcers (1).
FIG. 1. Molecular structure of Cbx.
With prolonged treatment, Cbx produces a pseudohyperaldosteronism by binding to mineralocorticoid receptors and inhibiting 11?-hydroxysteroid dehydrogenase (11HSD) type 2 at the level of epithelial target tissues for aldosterone (2). Cbx also has central hypertensive effects, without affecting saline appetite, when injected into a lateral ventricle (3). Cbx can also produce local glucocorticoid excess in vascular smooth cells, reflecting lower 11HSD2 activity, and plays an adjuvant role in coronary vascular inflammatory response during high salt intake (4). Indeed, vascular rings preincubated with 11-dehydrocorticosterone and Cbx display a decreased contractile response, compared with those incubated with 11-dehydrocorticosterone alone (5). 11HSD2 has also been involved in the nongenomic effects of aldosterone in human arteries. Cortisol has no effect on intracellular pH, but when applied in the presence of Cbx, a dramatic increase in Na+-H+ exchanger activity was evident (6). The type 1 enzyme has also been involved in the onset of placental apoptosis and reduced fetal and placental weight (7). Cbx is also able to reduce intracellular coupling via gap-junctions and is used as a type of gap-junction blocker in human fibroblasts (8). In vivo studies have demonstrated that GE administration induces cell apoptosis in thymocytes and splenocytes, probably due to inhibition of its metabolizing enzyme 11HSD1 in the liver (9).
A prooxidant effect of GE and aldosterone, due to a direct effect at the level of mineralocorticoid receptors, was recently demonstrated in human mononuclear leukocytes (10). Indeed, other recent papers (11, 12) have shown that GE induces an oxidative stress in Ca2+ loaded rat liver mitochondria (RLM) with the result of inducing mitochondrial permeability transition (MPT), a process strictly associated with programmed cell death (for recent reviews see Refs. 12 , 13).
Considering that during appropriate therapy Cbx is entirely located in the liver, gastrointestinal tract, and blood (14, 15), the aim of the present study was to evaluate the effect of Cbx as a possible MPT inducer and identify the mechanism and target of its action on the liver mitochondrial membrane.
Materials and Methods
Chemicals
Cbx was purchased from Sigma (St. Louis, MO) and dissolved in absolute ethanol. All other reagents were of the highest purity commercially available.
Mitochondrial preparations
RLM were isolated by conventional differential centrifugation in a buffer containing 250 mM sucrose, 5 mM HEPES (pH 7.4), and 1 mM EGTA; EGTA was omitted from the final washing solution. Protein content was measured by the biuret method with BSA as a standard (16).
Standard incubation procedures
RLM (1 mg protein per milliliter) were incubated in a water-jacketed cell at 20 C. The standard medium contained 200 mM sucrose, 10 mM HEPES (pH 7.4), 5 mM succinate, and 1.25 μM rotenone. Variations and/or other additions are described with the individual experiments presented. The control assays contained the same volume of ethanol as those carried out with Cbx; the final ethanol concentration did not exceed 0.1% (vol/vol) and did not affect the assayed activities.
Determination of mitochondrial functions
Membrane potential () was calculated on the basis of distribution of the lipid-soluble cation tetraphenylphosphonium through the inner membrane, measured using a tetraphenylphosphonium-specific electrode prepared in our laboratory according to published procedures (17).
Mitochondrial swelling was determined by measuring the apparent absorbance change of mitochondrial suspensions at 540 nm using a Uvikon model 922 spectrophotometer (Kontron, Eching, Germany) equipped with thermostatic control.
The protein sulfhydryl group oxidation assay was performed as in Santos et al. (18).
The production of H2O2 in RLM was measured fluorometrically by the scopoletin method (19) in a 4-8202 spectrofluorometer (Aminco-Bowman, Silver Spring, MD).
Oxygen uptake was measured with the Clark electrode. The redox state of endogenous pyridine nucleotides was followed fluorometrically in an Aminco-Bowman 4-8202 spectrofluorometer with excitation at 354 nm and emission at 462 nm.
Detection of cytochrome c release
RLM (1 mg protein per milliliter) were incubated for 15 min at 20 C in the standard medium with the appropriate additions. The reaction mixtures were then centrifuged at 13,000 x g for 10 min at 4 C to obtain mitochondrial pellets. The supernatant fractions were further spun at 100,000 x g for 15 min at 4 C to eliminate mitochondrial membrane fragments and concentrated five times by ultrafiltration through Centrikon 10 membranes (Amicon, Beverly, MA) at 4 C. Aliquots of the concentrated supernatants were subjected to 15% SDS-PAGE for cytochrome c and analyzed by Western blotting using a mouse anticytochrome c antibody (PharMingen, San Diego, CA).
Results
The addition of 10 μM Cbx to RLM suspension, incubated for 15 min in standard medium in the presence of 40 μM Ca2+, induces a decrease in the apparent absorbance at 540 nm of approximately 0.3 U (Fig. 2A), indicative of mitochondrial swelling. This extent of swelling is far lower than that previously observed with GE (11). Mitochondrial swelling induced by Cbx is paralleled by a collapse of (Fig. 2B). The effects of Cbx on mitochondrial permeability are strictly Ca2+ dependent. This is demonstrated by the complete inefficacy exhibited by the drug in the absence of Ca2+ and the total block of both swelling induction and drop (Fig. 2, A and B, respectively) by ruthenium red, a well-known inhibitor of Ca2+ transport. Furthermore, it is noteworthy that swelling induction and drop are almost completely blocked by cyclosporin A (CsA), which interacts with mitochondrial cyclophylin; bongkrekic acid (BKA), an inhibitor of adenine nucleotide translocase; or N-ethylmaleimide (NEM), a thiol alkylating agent (Fig. 2, A and B). Instead, an inhibitor of reactive oxygen species (ROS), catalase, exhibits a slight inhibition (Fig. 2, A and B). The inset of Fig. 2A reports the dose-dependent effect of Cbx (S0.5 = 12.2 μM) on mitochondrial swelling after 15 min. The swelling of RLM and the drop in their are accompanied by enhanced oxygen uptake, which is observable, although to a lesser extent, also in the presence of Ca2+ (Fig. 3A).
FIG. 2. Mitochondrial swelling (A) and collapse (B) induced by Cbx in RLM. RLM were incubated in standard medium supplemented with 40 μM Ca2+ under the conditions indicated in Materials and Methods. Cbx was present at a concentration of 10 μM. Where indicated, 1 μM CsA, 5 μM BKA, 10 μM NEM, 1000 U/mg catalase (cat), or 1 μM ruthenium red (RR) was present. A downward deflection (A) indicates mitochondrial swelling. The inset (A) shows the dose-dependent effect of Cbx calculated as the maximum extent of A after 15 min of incubation at every concentration. The E value (B) refers to the electrode potential. Four other assays gave comparable results.
FIG. 3. Oxygen uptake increase (A) and hydrogen peroxide generation (B) induced by Cbx in the absence and presence of Ca2+. RLM were incubated in standard medium in the presence of 10 μM Cbx and 40 μM Ca2+ under the conditions described in Materials and Methods. A (arrows), 50 nM antimycin A (Ant A) and 1000 U/mg protein catalase (cat) were added. The assays were performed four times with almost identical results. B, Mean values ± SD of four experiments are reported.
The addition of the respiratory chain inhibitor, antimycin A, and catalase, results in O2 production, which is evidenced by the reversed trend of oxygen uptake traces, indicating H2O2 generation in both conditions (Fig. 3A). The results in Fig. 3B give direct confirmation of H2O2 generation by Cbx, which is approximately 0.8 nmol/mg protein with Ca2+, lower than that obtainable with GE (20), and 0.2 nmol/mg protein in the absence of the cation.
These preliminary observations show that Cbx, in the absence of Ca2+, produces an increase in membrane permeability, most likely by means of an oxidative stress. To confirm this hypothesis, we investigated the effect of Cbx in inducing pyridine nucleotide and protein sulfhydryl group oxidation. The results in Fig. 4A show that Cbx, in the presence of Ca2+, induces pyridine nucleotide oxidation, which is prevented by CsA, BKA, and NEM. In the absence of Ca2+, the effect of Cbx is negligible (Fig. 4A). The redox state of mitochondrial thiol groups is reported in Fig. 4B: Cbx, in the presence of Ca2+, is able to oxidize approximately 16% of the total thiols, whereas alone it oxidizes only about 4%.
FIG. 4. Pyridine nucleotide (A) and sulfhydryl group (B) oxidation in the presence of Cbx. RLM were incubated as described in the legend to Fig. 3A. Where indicated, 1 μM CsA, 5 μM BKA, or 10 μM NEM were present. A, Results from a typical experiment. Four other experiments gave identical results. NAD+(P), Nicotinamide adenine dinucleotide phosphate (oxidized form); NAD(P)H, nicotinamide adenine dinucleotide phosphate reduced. B, Mean values of four experiments are reported. The total content of reduced sulfhydryl groups was 95 nmol/mgprotein.
The observation that Cbx is able to induce mitochondrial swelling suggests that the outer membrane might undergo some rupture with loss of mitochondrial proteins from the intermembrane space.
The Western blot analysis presented in Fig. 5 demonstrates that RLM incubated in the presence of Cbx and Ca2+ release the proapoptotic factor cytochrome c, suggesting that Cbx can be considered as a potential proapoptotic agent. The release of the proapoptotic factor is inhibited by CsA and NEM.
FIG. 5. Cbx induces the release of cytochrome c in RLM. RLM were incubated for 15 min in standard medium supplemented with 40 μM Ca2+. Where indicated, 10 μM Cbx, 1 μM CsA, or 10 μM NEM were present. The incubation was followed by centrifugation and recovery of supernatants, which were 5-fold concentrate, subjected to SDS-PAGE and immunoblotting to detect cytochrome c as described in Materials and Methods. The data shown are typical of three separate experiments.
To gain information about the site(s) of action of Cbx on the mitochondrial membrane, we examined the possibility of its interaction with some transition metals of the respiratory chain, in the oxidized state, using the same approach used for isoflavonoids (21) and triterpenoids (20). The basis of this strategy is to induce different redox levels on the respiratory complexes to identify their involvement in the interaction. The results in Fig. 6 show that RLM, incubated with Cbx in the presence of ?-hydroxybutyrate as the energizing substrate, which donates electrons to complex I, exhibit a large amplitude swelling (Fig. 6A, Cbx) accompanied by a rapid and complete drop (Fig. 6B, Cbx). The addition to the medium of the inhibitor rotenone, which blocks ?-hydroxybutyrate oxidation, and succinate, which restores electron flux at the level of complex II, produces a remarkable inhibition of mitochondrial swelling induced by Cbx (Fig. 6C, Cbx), whereas undergoes only a slight transient drop (Fig. 6D, Cbx).
FIG. 6. Effect of different redox states of the respiratory chain on the mitochondrial swelling (A, C, and E) and collapse (B, D, and F) induced by Cbx. All the incubations were carried out as indicated in Materials and Methods in the presence of 200 mM sucrose, 10 mM HEPES (pH 7.4), 40 μM Ca2+, and 5 mM ?-hydroxybutyrate (?OH). C–F, 5 mM succinate (Succ) and 1.25 μM rotenone (Rot) were present in the experiments. E and F, 3 mM ascorbate (Asc), 100 μM TMPD, and 50 nM antimycin A (Ant A) were present in the experiments. Where indicated, 10 μM Cbx were present. In the swelling experiments (A, C, and E), all the compounds were present in the medium; in determinations, the compounds were added in the indicated sequences.
A different picture occurs when RLM are energized with ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), which donate electrons to cytochrome c, and are again incubated in the presence of ?-hydroxybutyrate plus rotenone and succinate plus antimycin A, which maintain the reduced state of complex II and part of complex III (until the UQ–/bH heme couple at the Qn site) (22). In these experimental conditions (Fig. 6E, Cbx), the inhibition of swelling is less marked and is accompanied by a gradual but incomplete drop (Fig. 6F, Cbx). It is worth noting that in the measurements of reported in Fig. 6, unlike the other experiments, all the compounds have been added in a particular sequence to demonstrate the different energized state of RLM on each addition. These experiments show that the MPT induced by Cbx is nearly entirely prevented when the electron transporters of complex I are in a fully reduced state. However, the reduction of some component of complex II and III can also be involved in the inhibition of the phenomenon.
Discussion
Our study shows that incubation of RLM with 10 μM Cbx results in a dose-dependent matrix swelling and collapse (Fig. 2, A and B). This concentration of Cbx has been used by other authors, as reported in a very recent investigation regarding the effect of the drug on the activity of 11HSD1 in hepatocytes, liver slices, and homogenates (23). The reported experiments demonstrate that mitochondria of the mentioned preparations can be exposed to Cbx concentrations of nearly 10 μM. Cbx behaves as a typical inducer of MPT and, due to the release of cytochrome c (Fig. 5), can be considered a potential proapoptotic agent. Furthermore, it should also be emphasized that Cbx induces pore opening by means of an oxidative stress as the consequence of its interaction with the electron transport chain. Cbx, in fact, is able to produce ROS, as indirectly demonstrated by the increase in both oxygen uptake and oxygen production on addition of antimycin A plus catalase (Fig. 3A) and directly by the production of H2O2 (Fig. 3B). The above-mentioned oxidant effects are the result of the direct interaction of Cbx with the respiratory chain in the absence of Ca2+ and are not related to the opening of the transition pore, which takes place only in the presence of Ca2+. The reduced swelling observed, if compared with that induced by GE (11), is most likely due to the lower production of H2O2 (20), as result of the presence of the succinate moiety in the molecule of Cbx (Fig. 1). This charged group, by rendering the molecule more hydrophilic (15), would be disadvantageous for Cbx in reaching its site(s) of action in the bulk of the mitochondrial inner membrane. Indeed, it should be emphasized that in liver the succinate moiety is not hydrolyzed to GE and succinate as happens in other tissues. Furthermore, no metabolic transformation of the triterpenoid moiety of Cbx has been detected in man or animals (15).
A possible mechanism for MPT induction, as also previously suggested (24), is that Cbx produces H2O2 in the absence of Ca2+ (Fig. 3, A and B). This ROS oxidizes a very small amount of thiol groups (Fig. 4B), which, however, are critical to inducing the opening of the pore when Ca2+ is present (Fig. 2, A and B). The increase in oxygen uptake leads to an increase in both hydrogen peroxide generation (Fig. 3B) and thiol group oxidation (Fig. 4B) as well as the oxidation of endogenous pyridine nucleotides (Fig. 4A).
The reactive site of Cbx, as also proposed for GE (20) and other compounds structurally related to uric acid (25), is the conjugated carbonyl oxygen at C-11, which can be involved in keto-enol tautomerism. This group would interact with a transition metal, most likely an oxidized form of iron, Fe3+, thus producing a Cbx-derived oxygen radical by means of the following reaction as also proposed elsewhere (25).
The implication of ROS in inducing the MPT is suggested by the results of Fig. 2, A and B, which show that catalase is able to induce an inhibition of the phenomenon. The incomplete inhibition is likely due to the difficulty of hydrogen peroxide in diffusing outwardly from the site of its production. Indeed, this result suggests that H2O2 can directly oxidize a part of the critical thiol groups via the glutathione peroxidase/glutathione reductase system.
The results reported in Fig. 6 identify the component of the electron transport chain involved in the interaction with Cbx. It should be pointed out that the strategy described requires particular experimental conditions different from those generally used in this kind of investigation (presence of succinate plus rotenone to energize RLM).
In this regard, the maximal extent of swelling and the most rapid collapse was obtained during the oxidation of ?-hydroxybutyrate (Fig. 6, A and B, respectively). These effects are strongly inhibited when rotenone is also added together with succinate (Fig. 6, C and D). In this condition the components of complex I are completely reduced, suggesting that an Fe3+ of some Fe-S center belonging to this complex is the main component responsible for the phenomenon. In fact, if we consider the redox couple of Cbx to be of the ubiquinol/semiquinone type, whose normal redox potential is about –160 mV (25), it is possible that the Fe-S center involved is the so-called N-2 center, which has a normal redox potential in the range of –20 to –160 mV (26). The incomplete inhibition observed, however, raises the hypothesis that some other redox couple might also be involved in the phenomenon, although to a much lesser extent. To identify this component, RLM were treated with ascorbate plus TMPD in addition to ?-hydroxybutyrate, rotenone, and succinate. The results obtained in this condition, showing a less effective MPT inhibition than previously (compare Fig. 6, E and F, with Fig. 6, C and D), are consistent with the involvement of an oxidized component located upward from the site of antimycin A inhibition.
In fact, Fe-S of Riske protein and Fe3+ of cytochrome c1 must be excluded because their redox potential is almost identical with that of cytochrome c; they are thus completely reduced by a reversal of the electron flux that also progresses back up the chain. Some component of complex IV in an oxidized state could be involved, but the well-known defense mechanism against ROS production present in this complex excludes this possibility. One proposal is that in the presence of antimycin A, there is an accumulation of the semiquinone radical UQ–, which autooxidizes in the presence of molecular oxygen; this leads to the generation of superoxide anion and other ROS (27). However, because Cbx does not remove the inhibition of complex I due to rotenone (result not reported), part of the enhanced oxygen uptake by Cbx observed in the absence of Ca2+ (Fig. 3A) might be explained by an accumulation of semiquinone radical that is favored by Cbx and that increases electron flux along complexes II and III, although it should be pointed out that another part of this oxygen uptake increase is also due to H2O2 generation at the level of the N-2 site of complex I as explained above.
Recently Alzamora et al. (6) have shown that cortisol produces a dramatic increase in Na+/H+ exchange activity in the presence of Cbx in human arteries, which is not reversed by spironolactone. Incubations with cortisol alone did not produce any effect. These findings suggest that vascular 11HSD plays an active role in maintaining the specificity of the rapid effects of aldosterone (6). From these data we can speculate that the overlapping of genomic and nongenomic effects can produce the complex properties of Cbx, both in vitro and in vivo.
In conclusion, our results support the hypothesis that in rat the in vivo proapoptotic effect of Cbx is due not to only the inhibition of 11HSD, which enhances corticosterone availability in the cells (28), but also MPT induction, thus indicating a toxicological effect of Cbx at the mitochondria level. It should also be emphasized that a relationship might exist in vivo between the effect of Cbx on 11HSD and MPT induction. As recently reported, Cbx. by inhibiting this enzyme. provokes an increase in the production of 7-ketocholesterol (7-KC) in liver (23). Among its many effects, 7-KC induces an increase in cytosolic Ca2+ concentrations in liver cells (29), thereby provoking favorable conditions for the induction of MPT in vivo and consequently apoptosis. In fact, in these conditions RLM are exposed to Cbx and high Ca2+ concentrations. The observation that 7-KC is also able to provoke apoptosis (23, 30) strongly supports this proposal. These combined effects of Cbx can explain its positive influence on cognitive function and its antiinflammatory and antiviral properties. Indeed, because apoptosis is also considered a safety mechanism activated by organisms to eliminate abnormal or damaged cells, the observed MPT induction can account for Cbx’s anticarcinogenic effects.
The final effect in clinical pathology depends on the particular characteristics of the cells in specific diseases. For example, some cancer mitochondria exhibit an overexpression of hexokinase, a protein that facilitates the opening of the transition pore and favors the action of the MPT inducers (31). In this regard, the above-mentioned effect of Cbx observed in isolated RLM, although reduced when compared with GE, can assume great importance in vivo in liver, in which the drug, by maintaining the succinate moiety, can act at the level of the cells susceptible to MPT without affecting normal cells.
Acknowledgments
The authors are grateful to Mr. Mario Mancon for his skilled technical assistance and Mr. Martin Donach for his help in the preparation of the manuscript.
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Address all correspondence and requests for reprints to: Professor Antonio Toninello, Dipartimento di Chimica Biologica, Università di Padova, Istituto di Neuroscienze del Consiglio Nazionale delle Ricerche, Unità per lo Studio delle Biomembrane, viale G. Colombo 3, 35121 Padova, Italy. E-mail: antonio.toninello@unipd.it.
Abstract
Carbenoxolone (Cbx), a derivative of glycyrrhetinic acid, which has been found to affect mineralocorticoid and glucocorticoid receptors, induces swelling and membrane potential collapse when added to Ca2+-loaded liver mitochondria at 10 μM concentrations.
These effects are strictly correlated with hydrogen peroxide generation, increase in oxygen uptake, and sulfhydryl and pyridine nucleotide oxidation. Cyclosporin A, bongkrekic acid, and N-ethylmaleimide completely abolish all the above-described effects, suggesting that Cbx can be considered an inducer of mitochondrial permeability transition by means of oxidative stress. Cbx can also trigger the apoptotic pathway because the above events are also correlated with the loss of cytochrome c. These effects are probably related to the conjugated carbonyl oxygen in C-11, which produces reactive oxygen species by interacting with the mitochondrial respiratory chain, mainly at the level of complex I but, most likely, also with complex III. The oxidative stress induced by Cbx, which is responsible for pore opening, excludes that this is related to a genomic effect of the compound.
Introduction
CARBENOXOLONE (CBX) IS the 3-hemisuccinate of glycyrrhetinic acid (GE), the active principle of licorice (for structure see Fig. 1); it has been used for 40 yr in the treatment of gastritis and ulcers (1).
FIG. 1. Molecular structure of Cbx.
With prolonged treatment, Cbx produces a pseudohyperaldosteronism by binding to mineralocorticoid receptors and inhibiting 11?-hydroxysteroid dehydrogenase (11HSD) type 2 at the level of epithelial target tissues for aldosterone (2). Cbx also has central hypertensive effects, without affecting saline appetite, when injected into a lateral ventricle (3). Cbx can also produce local glucocorticoid excess in vascular smooth cells, reflecting lower 11HSD2 activity, and plays an adjuvant role in coronary vascular inflammatory response during high salt intake (4). Indeed, vascular rings preincubated with 11-dehydrocorticosterone and Cbx display a decreased contractile response, compared with those incubated with 11-dehydrocorticosterone alone (5). 11HSD2 has also been involved in the nongenomic effects of aldosterone in human arteries. Cortisol has no effect on intracellular pH, but when applied in the presence of Cbx, a dramatic increase in Na+-H+ exchanger activity was evident (6). The type 1 enzyme has also been involved in the onset of placental apoptosis and reduced fetal and placental weight (7). Cbx is also able to reduce intracellular coupling via gap-junctions and is used as a type of gap-junction blocker in human fibroblasts (8). In vivo studies have demonstrated that GE administration induces cell apoptosis in thymocytes and splenocytes, probably due to inhibition of its metabolizing enzyme 11HSD1 in the liver (9).
A prooxidant effect of GE and aldosterone, due to a direct effect at the level of mineralocorticoid receptors, was recently demonstrated in human mononuclear leukocytes (10). Indeed, other recent papers (11, 12) have shown that GE induces an oxidative stress in Ca2+ loaded rat liver mitochondria (RLM) with the result of inducing mitochondrial permeability transition (MPT), a process strictly associated with programmed cell death (for recent reviews see Refs. 12 , 13).
Considering that during appropriate therapy Cbx is entirely located in the liver, gastrointestinal tract, and blood (14, 15), the aim of the present study was to evaluate the effect of Cbx as a possible MPT inducer and identify the mechanism and target of its action on the liver mitochondrial membrane.
Materials and Methods
Chemicals
Cbx was purchased from Sigma (St. Louis, MO) and dissolved in absolute ethanol. All other reagents were of the highest purity commercially available.
Mitochondrial preparations
RLM were isolated by conventional differential centrifugation in a buffer containing 250 mM sucrose, 5 mM HEPES (pH 7.4), and 1 mM EGTA; EGTA was omitted from the final washing solution. Protein content was measured by the biuret method with BSA as a standard (16).
Standard incubation procedures
RLM (1 mg protein per milliliter) were incubated in a water-jacketed cell at 20 C. The standard medium contained 200 mM sucrose, 10 mM HEPES (pH 7.4), 5 mM succinate, and 1.25 μM rotenone. Variations and/or other additions are described with the individual experiments presented. The control assays contained the same volume of ethanol as those carried out with Cbx; the final ethanol concentration did not exceed 0.1% (vol/vol) and did not affect the assayed activities.
Determination of mitochondrial functions
Membrane potential () was calculated on the basis of distribution of the lipid-soluble cation tetraphenylphosphonium through the inner membrane, measured using a tetraphenylphosphonium-specific electrode prepared in our laboratory according to published procedures (17).
Mitochondrial swelling was determined by measuring the apparent absorbance change of mitochondrial suspensions at 540 nm using a Uvikon model 922 spectrophotometer (Kontron, Eching, Germany) equipped with thermostatic control.
The protein sulfhydryl group oxidation assay was performed as in Santos et al. (18).
The production of H2O2 in RLM was measured fluorometrically by the scopoletin method (19) in a 4-8202 spectrofluorometer (Aminco-Bowman, Silver Spring, MD).
Oxygen uptake was measured with the Clark electrode. The redox state of endogenous pyridine nucleotides was followed fluorometrically in an Aminco-Bowman 4-8202 spectrofluorometer with excitation at 354 nm and emission at 462 nm.
Detection of cytochrome c release
RLM (1 mg protein per milliliter) were incubated for 15 min at 20 C in the standard medium with the appropriate additions. The reaction mixtures were then centrifuged at 13,000 x g for 10 min at 4 C to obtain mitochondrial pellets. The supernatant fractions were further spun at 100,000 x g for 15 min at 4 C to eliminate mitochondrial membrane fragments and concentrated five times by ultrafiltration through Centrikon 10 membranes (Amicon, Beverly, MA) at 4 C. Aliquots of the concentrated supernatants were subjected to 15% SDS-PAGE for cytochrome c and analyzed by Western blotting using a mouse anticytochrome c antibody (PharMingen, San Diego, CA).
Results
The addition of 10 μM Cbx to RLM suspension, incubated for 15 min in standard medium in the presence of 40 μM Ca2+, induces a decrease in the apparent absorbance at 540 nm of approximately 0.3 U (Fig. 2A), indicative of mitochondrial swelling. This extent of swelling is far lower than that previously observed with GE (11). Mitochondrial swelling induced by Cbx is paralleled by a collapse of (Fig. 2B). The effects of Cbx on mitochondrial permeability are strictly Ca2+ dependent. This is demonstrated by the complete inefficacy exhibited by the drug in the absence of Ca2+ and the total block of both swelling induction and drop (Fig. 2, A and B, respectively) by ruthenium red, a well-known inhibitor of Ca2+ transport. Furthermore, it is noteworthy that swelling induction and drop are almost completely blocked by cyclosporin A (CsA), which interacts with mitochondrial cyclophylin; bongkrekic acid (BKA), an inhibitor of adenine nucleotide translocase; or N-ethylmaleimide (NEM), a thiol alkylating agent (Fig. 2, A and B). Instead, an inhibitor of reactive oxygen species (ROS), catalase, exhibits a slight inhibition (Fig. 2, A and B). The inset of Fig. 2A reports the dose-dependent effect of Cbx (S0.5 = 12.2 μM) on mitochondrial swelling after 15 min. The swelling of RLM and the drop in their are accompanied by enhanced oxygen uptake, which is observable, although to a lesser extent, also in the presence of Ca2+ (Fig. 3A).
FIG. 2. Mitochondrial swelling (A) and collapse (B) induced by Cbx in RLM. RLM were incubated in standard medium supplemented with 40 μM Ca2+ under the conditions indicated in Materials and Methods. Cbx was present at a concentration of 10 μM. Where indicated, 1 μM CsA, 5 μM BKA, 10 μM NEM, 1000 U/mg catalase (cat), or 1 μM ruthenium red (RR) was present. A downward deflection (A) indicates mitochondrial swelling. The inset (A) shows the dose-dependent effect of Cbx calculated as the maximum extent of A after 15 min of incubation at every concentration. The E value (B) refers to the electrode potential. Four other assays gave comparable results.
FIG. 3. Oxygen uptake increase (A) and hydrogen peroxide generation (B) induced by Cbx in the absence and presence of Ca2+. RLM were incubated in standard medium in the presence of 10 μM Cbx and 40 μM Ca2+ under the conditions described in Materials and Methods. A (arrows), 50 nM antimycin A (Ant A) and 1000 U/mg protein catalase (cat) were added. The assays were performed four times with almost identical results. B, Mean values ± SD of four experiments are reported.
The addition of the respiratory chain inhibitor, antimycin A, and catalase, results in O2 production, which is evidenced by the reversed trend of oxygen uptake traces, indicating H2O2 generation in both conditions (Fig. 3A). The results in Fig. 3B give direct confirmation of H2O2 generation by Cbx, which is approximately 0.8 nmol/mg protein with Ca2+, lower than that obtainable with GE (20), and 0.2 nmol/mg protein in the absence of the cation.
These preliminary observations show that Cbx, in the absence of Ca2+, produces an increase in membrane permeability, most likely by means of an oxidative stress. To confirm this hypothesis, we investigated the effect of Cbx in inducing pyridine nucleotide and protein sulfhydryl group oxidation. The results in Fig. 4A show that Cbx, in the presence of Ca2+, induces pyridine nucleotide oxidation, which is prevented by CsA, BKA, and NEM. In the absence of Ca2+, the effect of Cbx is negligible (Fig. 4A). The redox state of mitochondrial thiol groups is reported in Fig. 4B: Cbx, in the presence of Ca2+, is able to oxidize approximately 16% of the total thiols, whereas alone it oxidizes only about 4%.
FIG. 4. Pyridine nucleotide (A) and sulfhydryl group (B) oxidation in the presence of Cbx. RLM were incubated as described in the legend to Fig. 3A. Where indicated, 1 μM CsA, 5 μM BKA, or 10 μM NEM were present. A, Results from a typical experiment. Four other experiments gave identical results. NAD+(P), Nicotinamide adenine dinucleotide phosphate (oxidized form); NAD(P)H, nicotinamide adenine dinucleotide phosphate reduced. B, Mean values of four experiments are reported. The total content of reduced sulfhydryl groups was 95 nmol/mgprotein.
The observation that Cbx is able to induce mitochondrial swelling suggests that the outer membrane might undergo some rupture with loss of mitochondrial proteins from the intermembrane space.
The Western blot analysis presented in Fig. 5 demonstrates that RLM incubated in the presence of Cbx and Ca2+ release the proapoptotic factor cytochrome c, suggesting that Cbx can be considered as a potential proapoptotic agent. The release of the proapoptotic factor is inhibited by CsA and NEM.
FIG. 5. Cbx induces the release of cytochrome c in RLM. RLM were incubated for 15 min in standard medium supplemented with 40 μM Ca2+. Where indicated, 10 μM Cbx, 1 μM CsA, or 10 μM NEM were present. The incubation was followed by centrifugation and recovery of supernatants, which were 5-fold concentrate, subjected to SDS-PAGE and immunoblotting to detect cytochrome c as described in Materials and Methods. The data shown are typical of three separate experiments.
To gain information about the site(s) of action of Cbx on the mitochondrial membrane, we examined the possibility of its interaction with some transition metals of the respiratory chain, in the oxidized state, using the same approach used for isoflavonoids (21) and triterpenoids (20). The basis of this strategy is to induce different redox levels on the respiratory complexes to identify their involvement in the interaction. The results in Fig. 6 show that RLM, incubated with Cbx in the presence of ?-hydroxybutyrate as the energizing substrate, which donates electrons to complex I, exhibit a large amplitude swelling (Fig. 6A, Cbx) accompanied by a rapid and complete drop (Fig. 6B, Cbx). The addition to the medium of the inhibitor rotenone, which blocks ?-hydroxybutyrate oxidation, and succinate, which restores electron flux at the level of complex II, produces a remarkable inhibition of mitochondrial swelling induced by Cbx (Fig. 6C, Cbx), whereas undergoes only a slight transient drop (Fig. 6D, Cbx).
FIG. 6. Effect of different redox states of the respiratory chain on the mitochondrial swelling (A, C, and E) and collapse (B, D, and F) induced by Cbx. All the incubations were carried out as indicated in Materials and Methods in the presence of 200 mM sucrose, 10 mM HEPES (pH 7.4), 40 μM Ca2+, and 5 mM ?-hydroxybutyrate (?OH). C–F, 5 mM succinate (Succ) and 1.25 μM rotenone (Rot) were present in the experiments. E and F, 3 mM ascorbate (Asc), 100 μM TMPD, and 50 nM antimycin A (Ant A) were present in the experiments. Where indicated, 10 μM Cbx were present. In the swelling experiments (A, C, and E), all the compounds were present in the medium; in determinations, the compounds were added in the indicated sequences.
A different picture occurs when RLM are energized with ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), which donate electrons to cytochrome c, and are again incubated in the presence of ?-hydroxybutyrate plus rotenone and succinate plus antimycin A, which maintain the reduced state of complex II and part of complex III (until the UQ–/bH heme couple at the Qn site) (22). In these experimental conditions (Fig. 6E, Cbx), the inhibition of swelling is less marked and is accompanied by a gradual but incomplete drop (Fig. 6F, Cbx). It is worth noting that in the measurements of reported in Fig. 6, unlike the other experiments, all the compounds have been added in a particular sequence to demonstrate the different energized state of RLM on each addition. These experiments show that the MPT induced by Cbx is nearly entirely prevented when the electron transporters of complex I are in a fully reduced state. However, the reduction of some component of complex II and III can also be involved in the inhibition of the phenomenon.
Discussion
Our study shows that incubation of RLM with 10 μM Cbx results in a dose-dependent matrix swelling and collapse (Fig. 2, A and B). This concentration of Cbx has been used by other authors, as reported in a very recent investigation regarding the effect of the drug on the activity of 11HSD1 in hepatocytes, liver slices, and homogenates (23). The reported experiments demonstrate that mitochondria of the mentioned preparations can be exposed to Cbx concentrations of nearly 10 μM. Cbx behaves as a typical inducer of MPT and, due to the release of cytochrome c (Fig. 5), can be considered a potential proapoptotic agent. Furthermore, it should also be emphasized that Cbx induces pore opening by means of an oxidative stress as the consequence of its interaction with the electron transport chain. Cbx, in fact, is able to produce ROS, as indirectly demonstrated by the increase in both oxygen uptake and oxygen production on addition of antimycin A plus catalase (Fig. 3A) and directly by the production of H2O2 (Fig. 3B). The above-mentioned oxidant effects are the result of the direct interaction of Cbx with the respiratory chain in the absence of Ca2+ and are not related to the opening of the transition pore, which takes place only in the presence of Ca2+. The reduced swelling observed, if compared with that induced by GE (11), is most likely due to the lower production of H2O2 (20), as result of the presence of the succinate moiety in the molecule of Cbx (Fig. 1). This charged group, by rendering the molecule more hydrophilic (15), would be disadvantageous for Cbx in reaching its site(s) of action in the bulk of the mitochondrial inner membrane. Indeed, it should be emphasized that in liver the succinate moiety is not hydrolyzed to GE and succinate as happens in other tissues. Furthermore, no metabolic transformation of the triterpenoid moiety of Cbx has been detected in man or animals (15).
A possible mechanism for MPT induction, as also previously suggested (24), is that Cbx produces H2O2 in the absence of Ca2+ (Fig. 3, A and B). This ROS oxidizes a very small amount of thiol groups (Fig. 4B), which, however, are critical to inducing the opening of the pore when Ca2+ is present (Fig. 2, A and B). The increase in oxygen uptake leads to an increase in both hydrogen peroxide generation (Fig. 3B) and thiol group oxidation (Fig. 4B) as well as the oxidation of endogenous pyridine nucleotides (Fig. 4A).
The reactive site of Cbx, as also proposed for GE (20) and other compounds structurally related to uric acid (25), is the conjugated carbonyl oxygen at C-11, which can be involved in keto-enol tautomerism. This group would interact with a transition metal, most likely an oxidized form of iron, Fe3+, thus producing a Cbx-derived oxygen radical by means of the following reaction as also proposed elsewhere (25).
The implication of ROS in inducing the MPT is suggested by the results of Fig. 2, A and B, which show that catalase is able to induce an inhibition of the phenomenon. The incomplete inhibition is likely due to the difficulty of hydrogen peroxide in diffusing outwardly from the site of its production. Indeed, this result suggests that H2O2 can directly oxidize a part of the critical thiol groups via the glutathione peroxidase/glutathione reductase system.
The results reported in Fig. 6 identify the component of the electron transport chain involved in the interaction with Cbx. It should be pointed out that the strategy described requires particular experimental conditions different from those generally used in this kind of investigation (presence of succinate plus rotenone to energize RLM).
In this regard, the maximal extent of swelling and the most rapid collapse was obtained during the oxidation of ?-hydroxybutyrate (Fig. 6, A and B, respectively). These effects are strongly inhibited when rotenone is also added together with succinate (Fig. 6, C and D). In this condition the components of complex I are completely reduced, suggesting that an Fe3+ of some Fe-S center belonging to this complex is the main component responsible for the phenomenon. In fact, if we consider the redox couple of Cbx to be of the ubiquinol/semiquinone type, whose normal redox potential is about –160 mV (25), it is possible that the Fe-S center involved is the so-called N-2 center, which has a normal redox potential in the range of –20 to –160 mV (26). The incomplete inhibition observed, however, raises the hypothesis that some other redox couple might also be involved in the phenomenon, although to a much lesser extent. To identify this component, RLM were treated with ascorbate plus TMPD in addition to ?-hydroxybutyrate, rotenone, and succinate. The results obtained in this condition, showing a less effective MPT inhibition than previously (compare Fig. 6, E and F, with Fig. 6, C and D), are consistent with the involvement of an oxidized component located upward from the site of antimycin A inhibition.
In fact, Fe-S of Riske protein and Fe3+ of cytochrome c1 must be excluded because their redox potential is almost identical with that of cytochrome c; they are thus completely reduced by a reversal of the electron flux that also progresses back up the chain. Some component of complex IV in an oxidized state could be involved, but the well-known defense mechanism against ROS production present in this complex excludes this possibility. One proposal is that in the presence of antimycin A, there is an accumulation of the semiquinone radical UQ–, which autooxidizes in the presence of molecular oxygen; this leads to the generation of superoxide anion and other ROS (27). However, because Cbx does not remove the inhibition of complex I due to rotenone (result not reported), part of the enhanced oxygen uptake by Cbx observed in the absence of Ca2+ (Fig. 3A) might be explained by an accumulation of semiquinone radical that is favored by Cbx and that increases electron flux along complexes II and III, although it should be pointed out that another part of this oxygen uptake increase is also due to H2O2 generation at the level of the N-2 site of complex I as explained above.
Recently Alzamora et al. (6) have shown that cortisol produces a dramatic increase in Na+/H+ exchange activity in the presence of Cbx in human arteries, which is not reversed by spironolactone. Incubations with cortisol alone did not produce any effect. These findings suggest that vascular 11HSD plays an active role in maintaining the specificity of the rapid effects of aldosterone (6). From these data we can speculate that the overlapping of genomic and nongenomic effects can produce the complex properties of Cbx, both in vitro and in vivo.
In conclusion, our results support the hypothesis that in rat the in vivo proapoptotic effect of Cbx is due not to only the inhibition of 11HSD, which enhances corticosterone availability in the cells (28), but also MPT induction, thus indicating a toxicological effect of Cbx at the mitochondria level. It should also be emphasized that a relationship might exist in vivo between the effect of Cbx on 11HSD and MPT induction. As recently reported, Cbx. by inhibiting this enzyme. provokes an increase in the production of 7-ketocholesterol (7-KC) in liver (23). Among its many effects, 7-KC induces an increase in cytosolic Ca2+ concentrations in liver cells (29), thereby provoking favorable conditions for the induction of MPT in vivo and consequently apoptosis. In fact, in these conditions RLM are exposed to Cbx and high Ca2+ concentrations. The observation that 7-KC is also able to provoke apoptosis (23, 30) strongly supports this proposal. These combined effects of Cbx can explain its positive influence on cognitive function and its antiinflammatory and antiviral properties. Indeed, because apoptosis is also considered a safety mechanism activated by organisms to eliminate abnormal or damaged cells, the observed MPT induction can account for Cbx’s anticarcinogenic effects.
The final effect in clinical pathology depends on the particular characteristics of the cells in specific diseases. For example, some cancer mitochondria exhibit an overexpression of hexokinase, a protein that facilitates the opening of the transition pore and favors the action of the MPT inducers (31). In this regard, the above-mentioned effect of Cbx observed in isolated RLM, although reduced when compared with GE, can assume great importance in vivo in liver, in which the drug, by maintaining the succinate moiety, can act at the level of the cells susceptible to MPT without affecting normal cells.
Acknowledgments
The authors are grateful to Mr. Mario Mancon for his skilled technical assistance and Mr. Martin Donach for his help in the preparation of the manuscript.
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