Inactivation of mitochondrial succinate dehydrogenase by adriamycin activated by horseradish peroxidase and hydrogen peroxide

Inactivation of mitochondrial succinate dehydrogenase by adriamycin activated by horseradish peroxidase and hydrogen peroxide

Chemico-Biological Interactions 145 (2003) 67 /75 www.elsevier.com/locate/chembioint Inactivation of mitochondrial succinate dehydrogenase by adriam...

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Chemico-Biological Interactions 145 (2003) 67 /75 www.elsevier.com/locate/chembioint

Inactivation of mitochondrial succinate dehydrogenase by adriamycin activated by horseradish peroxidase and hydrogen peroxide Sanae Muraoka, Toshiaki Miura * Department of Biochemistry, Hokkaido College of Pharmacy, Katsuraoka-cho 7-1, Otaru 047-0264, Japan Received 17 July 2002; accepted 12 October 2002

Abstract Although human cancers are widely treated with anthracycline drugs, these drugs have limited use because they are cardiotoxic. To clarify the cardiotoxic action of the anthracycline drug adriamycin (ADM), the inhibitory effect on succinate dehydrogenase (SDH) by ADM and other anthracyclines was examined by using pig heart submitochondrial particles. ADM rapidly inactivated mitochondrial SDH during its interaction with horseradish peroxidase (HRP) in the presence of H2O2 (HRP /H2O2). Butylated hydroxytoluene, iron-chelators, superoxide dismutase, mannitol and dimethylsulfoxide did not block the inactivation of SDH, indicating that lipid-derived radicals, iron /oxygen complexes, superoxide and hydroxyl radicals do not participate in SDH inactivation. Reduced glutathione was extremely efficient in blocking the enzyme inactivation, suggesting that the SH group in enzyme is very sensible to ADM activated by HRP /H2O2. Under anaerobic conditions, ADM with HRP /H2O2 caused inactivation of SDH, indicating that oxidized ADM directly attack the enzyme, which loses its activity. Other mitochondrial enzymes, including NADH dehydrogenase, NADH oxidase and cytochrome c oxidase, were little sensitive to ADM with HRP /H2O2. SDH was also sensitive to other anthracycline drugs except for aclarubicin. Mitochondrial creatine kinase (CK), which is attached to the outer face of the inner membrane of muscle mitochondria, was more sensitive to anthracyclines than SDH. SDH and CK were inactivated with loss of red color of anthracycline, indicating that oxidative activation of the B ring of anthracycline has a crucial role in inactivation of enzymes. Presumably, oxidative semiquinone or quinone produced from anthracyclines participates in the enzyme inactivation. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Adriamycin; Anthracycline; Creatine kinase; Horseradish peroxidase; Succinate dehydrogenase

1. Introduction

* Corresponding author. Tel.: /81-134-62-5111; fax: /81134-62-5161 E-mail address: [email protected] (T. Miura).

Anthracycline antibiotics, including adriamycin (ADM), are widely used to treat various human cancers [1,2], but their clinical use has been limited because they cause cumulative and dose-dependent cardiotoxicity [3,4]. Although several modes of

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action have been proposed, the exact molecular mechanisms of the biological activities of anthracycline drugs are not well understood. ADM is reduced to a semiquinone radical by NADPH-P450 reductase and NADH dehydrogenase [5,6]. In the presence of oxygen, the reductive semiquinone radical species produces superoxide and hydroxyl radicals, which cause peroxidation of unsaturated membrane lipids [7]. These events lead to disturbance of the membrane structure and dysfunction of mitochondria. The quinone-modified anthracycline drug 5iminodaunorubicin is readily oxidized by horseradish peroxidase (HRP) [8]. However, tissue damage by ADM oxidized by peroxidases remains to be clarified. We previously showed [9] that creatine kinase (CK), which is distributed abundantly in the heart and is associated with the physiological role of ATP generation in conjunction with the contractile or transport system [10], is strongly inactivated by ADM oxidized by HRP in the presence of H2O2 (HRP/H2O2). However, mitochondrial succinate dehydrogenase (SDH) is inhibited by ADM at extremely high concentrations [11]. These findings suggest that metabolism of anthracycline drugs by peroxidase and damage to mitochondrial enzymes contribute to cardiotoxicity induced by anthracycline drugs. ADM among anthracycline drugs has the most deleterious effect on the heart [12 /14]. We believe that a more exact biochemical understanding of the mitochondrial damage induced by oxidized anthracyclines is important. In this study, we show that pig heart mitochondrial enzymes are readily inactivated by ADM and other anthracyclines with HRP /H2O2.

2. Materials and methods 2.1. Materials ADM was obtained from Kyowa Hakko Co. Ltd, Tokyo, Japan; HRP was obtained from Wako Pure Chemical Industries, Co. Ltd. Osaka, Japan; epirubicin and idarubicin were obtained from Pharmacia Japan; pirarubicin was obtained from Meijiseika Co. Ltd; aclarubicin was obtained

from Yamanouchi Pharmaceutical Co. Ltd; daunorubicin, catalase (beef liver) and superoxide dismutase (bovine erythrocytes) were obtained from Sigma Chemical Co., St. Louis, MO. Other chemicals were highly analytical grade products obtained from commercial suppliers. 2.2. Preparation of submitochondrial particles (SMP) Pig heart mitochondria were prepared by the method of Crane et al. [15] with minor modifications. Pig heart muscle was minced, 10 mM Hepes buffer at pH 7.4 containing 0.25 M sucrose and 1.0 mM ethylenediamine tetraacetate (EDTA) was added, and then the mixture was homogenized by using a Waring blender homogenizer for 3 min at maximum rate at 4 8C. The homogenate (30% w/v) was centrifuged at 900 /g , and the supernatants were filtered through a double layer of gauze and then were centrifuged at 6000 /g to produce mitochondrial pellets. The pellets were further washed three times with 50 mM Hepes buffer at pH 7.4 and were stored at /80 8C. The mitochondria were suspended in 50 mM Hepes buffer and were sonicated for 40 s four times under argon gas. The unbroken mitochondria were removed by centrifuging at 8000 /g. The supernatant was used as a suspension of the SMP. The protein was measured by using the bicinchoninic acid assay that used bovine serum albumin as a standard [16]. 2.3. Reaction system SMP (0.1 mg protein/ml) were incubated for various times, with various concentrations of ADM in the presence of HRP /H2O2. All the incubations were performed at 37 8C in 50 mM Hepes buffer at pH 7.4 unless otherwise noted. For anaerobic experiments, the reaction mixture was purged with argon for 15 min, and the reactions were carried out under argon gas. 2.4. Measurement of enzyme activities The SDH activity was measured as the rate of 0.1 mM dichlorophenolindophenol reduction de-

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pendent on 3.0 mM succinate in the presence of 5.0 mM phenazine methosulfate (PMS) at 600 nm in 50 mM Hepes buffer [17]. The NADH oxidase activity was measured by the oxidation of 100 mM NADH at 340 nm and the NADH dehydrogenase activity was assayed by measuring the rate of NADH-dependent ferricyanide reduction at 420 nm [18]. The activity of cytochrome c oxidase was measured by the decrease in absorbance at 550 nm during oxidation of 50 mM reduced cytochrome c [19]. CK activity was measured at 30 8C by using a Wako Pure Chemical Industries CK kit. One unit of CK transferred 1.0 mmol of phosphate from phosphocreatine to ADP each minute at 30 8C. The activity of peroxidase was measured by the formation of I2 at 375 nm in 10 mM acetate buffer at pH 5.0 containing 0.27 mM H2O2, 1.7 mM KI and 25 nM HRP [20].

3. Results 3.1. Inactivation of SDH by ADM with HRP / H 2O 2

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Fig. 1. Loss of mitochondrial SDH activity by ADM activity by ADM activated by HRP /H2O2, SMP (0.1 mg protein/ml) was incubated with 10 mM ADM, 0.12 mM HRP and 100 mM H2O2 in 50 mM Hepes buffer. SDH activity was measured by the PMS-dichlorophenolindophenol assay as described in Section 2, SMP reduced dichlorophenolindophenol by 0.5 mmol/min/mg protein, where 1 mmol of dichlorophenolindophenol reduced is equivalent to 1 mmol oxidized succinate. After the incubation, an aliquot (0.2 ml) of the reaction mixture was used to measures SDH activity. Other conditions were described in Section 2. Each point represents the mean9/S.D. of five experiments. (k), /ADM; (m), without ADM and (^), without HRP /H2O2.

Fig. 1 shows that mitochondrial SDH was rapidly inactivated during interaction of ADM with HRP /H2O2. The inactivation was linear up to 10 min. After incubation for 30 min, about 80% of the enzyme activity was lost. Without HRP / H2O2 or ADM, the SDH activity slightly decreased. Fig. 2 shows that inactivation of SDH was dependent on the concentrations of ADM. The 50% inhibitory concentration (IC50) of ADM was about 0.8 mM. 3.2. Participation of free radicals Reactive oxygen species participate in lipid peroxidation of the mitochondrial membrane by ADM [7,21] or ADM /iron [22,23]. Lipid peroxidation inactivates mitochondrial enzymes in the respiratory chain [24]. We therefore examined if lipid peroxidation participates in inactivating SDH by ADM with HRP /H2O2 by using a strong antioxidant, butylated hydroxytoluene (BHT), and iron-chelators of EDTA and diethylenetriaminepentaacetic acid (DETAPAC). Adding the

Fig. 2. Effect of ADM concentrations on SDH activity. Conditions were the same as described in Fig. 1. After incubation for 30 min, SDH activity was measured. Each point represents the mean9/S.D. of five experiments.

antioxidant and iron-chelators did not block the inactivation of SDH by ADM with HRP /H2O2 (Table 1). Superoxide dismutase, dimethylsulfoxide and mannitol, which are scavengers of super-

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Table 1 Effect of radical scavengers and iron-chelators on SDH inactivation Additions

Concentration (mM)

SDH activity (%)

None None under anaerobic conditions

14.59/1.9 15.69/1.9

Superoxide dismutase 3 /10 4 3 /10 3

12.29/1.4 8.39/3.1

Catalase

4 /10 5 4 /10 4

83.89/10.4 89.49/8.7

BHT

0.1 0.5

13.99/4.7 16.49/3.3

EDTA

0.1 0.5

16.09/5.0 17.99/5.7

DETAPAC

0.1 0.5

14.69/5.3 16.59/5.3

Dimethylsulfoxide Mannitol Glutathione

100 100 0.1

13.89/3.6 14.79/5.1 95.79/4.0

Conditions were the same as described in Fig. 1 except for adding radical scavengers and iron-chelator. Anaerobic conditions were described in Section 2. Each value represents the mean9/S.D. of five experiments.

oxide and hydroxyl radicals, respectively, also had no effect. Catalase completely blocked SDH inactivation through removing H2O2. We did not detect thiobarbituric acid reactive substances formation in the SMP incubated with ADM and HRP /H2O2 (data not shown). Furthermore, under anaerobic conditions ADM with HRP /H2O2 caused inactivation of SDH. These results indicate that active oxygen species, such as superoxide and hydroxyl radicals, lipid-derived radicals and lipid peroxide, did not participate in the SDH inactivation, and that ADM activated by HRP /H2O2 directly attacked the enzyme to cause inactivation of SDH. Reduced glutathione (GSH) was extremely efficient in blocking the enzyme inactivation, suggesting that the SH group in the enzyme is very sensible to ADM activated by HRP /H2O2. 3.3. Inhibition of other enzymes in the mitochondrial respiratory chain Fig. 3 shows that other mitochondrial enzymes of the respiratory chain were inactivated by ADM

in a concentration dependently in the presence of HRP /H2O2. However, much more ADM was required to inactivate these respiratory enzymes than to inactivate SDH. The IC50 of NADH oxidase was about 30 mM, and NADH dehydrogenase and cytochrome c oxidase were only about 20 and 40%, respectively, inactivated at 50 mM ADM. These results indicate that SDH was much more sensitive to ADM activated by HRP /H2O2 than to the other respiratory enzymes. 3.4. Spectral change in anthracyclines and inactivation of SDH and CK Among anthracycline anticancer drugs, ADM is particularly toxic to heart [12 /14]. To clarify the cardiotoxicity of ADM, a relationship between metabolism of anthracycline drugs and inactivation of mitochondrial enzymes by HRP /H2O2 were examined. Fig. 4 shows the structures of anthracycline drugs used in this study. Fig. 5 shows spectral changes in anthracycline drugs, including ADM, epirubicin, idarubicin, pirarubicin, daunorubicin and aclarubicin, during the interaction with HRP /H2O2. Except for aclarubicin, anthracycline drugs used in this study were steadily metabolized and the red color of the drugs disappeared during the interaction with HRP /

Fig. 3. Loss of mitochondrial respiratory enzymes during the interaction of ADM with HRP /H2O2. Conditions were the same as described in Fig. 1 except for concentrations of ADM. Each point represents the mean9/S.D. of five experiments. (k), Cytochrome c oxidase; (m), NADH oxidase and (^), NADH dehydrogenase.

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Fig. 4. Structure of anthracycline drugs.

H2O2 for 3 min. ADM, epirubicin and idarubicin were more rapidly oxidized than other anthracyclines. The yellow color of aclarubicin was con-

stant during interaction with HRP /H2O2. Except for aclarubicin, which has a phenolic hydroxyl group in the B ring, all anthracyclines used in this

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Fig. 5. Spectral change in anthracycline drugs induced by HRP /H2O2. The reaction mixture contained 10 mM anthracyclines, 1.2 mM HRP and 100 mM H2O2 in 50 mM Hepes buffer. Spectral changes were recorded at 37 8C. The number of curves refer to time (min) of reaction.

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Table 2 SDH and CK inactivation by anthracycline drugs during interaction with HRP /H2O2 Additions

SDH CK Enzyme activity (%)

None Adriamycin Epirubicin Idarubicin Pirarubicin Daunomycin Acrarubicin

96.59/2.1 52.89/4.4 47.09/4.7 53.89/3.1 84.59/6.8 72.39/1.6 94.59/1.8

100.0 25.29/3.5 15.19/3.7 33.09/5.0 76.79/5.8 60.19/7.0 80.19/6.7

Conditions were the same as described in Fig. 5 except for adding SMP (0.1 mg protein/ml) to the reaction mixture. After incubation for 2 min on SDH and for 1 min on CK, respectively, activity of the enzymes was measured. Activity of CK was 1.13 u/ml with reaction mixture before incubation. Each value represents the mean9/S.D. of five experiments.

study have a p -hydroquinone structure in the B ring. Hydroquinone is easily oxidized by HRP / H2O2 [25]. Oxidation of the hydroquinone structure in the B ring should contribute to the disappearance of the red color of anthracyclines. We previously showed [9] that rabbit muscle CK is easily inactivated by ADM with HRP /H2O2. Also, CK is attached to the outer face of the inner membrane of the muscle mitochondria [26]. We examined the inactivation of SDH and CK with loss of red color of anthracycline. Table 2 summarizes that inactivation of mitochondrial SDH and CK were caused by anthracyclines with HRP / H2O2. Except for aclarubicin, anthracycline drugs readily inactivated mitochondrial SDH in the presence of HRP /H2O2. CK was more sensitive than SDH to anthracycline drugs with HRP / H2O2. ADM, epirubicin and idarubicin were more effective in inhibiting SDH and CK than pirarubicin and daunorubicin. Presumably, oxidative metabolites caused inactivation of the mitochondrial enzymes.

4. Discussion This study showed that ADM causes rapid inactivation of mitochondrial SDH in the presence of HRP /H2O2. ADM is reduced to a semiquinone form by complex I of the mitochondrial electron

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transfer chain [5,6] and by microsomal cytochrome P-450 reductase [27,28]. The redox cycling of the semiquinone radical leads to the formation of superoxide, H2O2 and hydroxyl radicals, and then lipid peroxidation is caused by these reactive oxygen species themselves or through reduction of iron by these reactive oxygen species [21 / 24,29,30]. However, the results of this study showed that ADM oxidized by HRP /H2O2 inactivates SDH and other mitochondrial enzymes of the respiratory chain. Superoxide or hydroxyl radicals inactivating SDH is unlikely because superoxide dismutase and mannitol, which are typical scavengers of superoxide and hydroxyl radicals, had no effect. Also, SDH was inactivated independently of lipid peroxidation because BHT, which stops lipid peroxidation, had no effect. Generally, iron has a crucial role in peroxidation reactions through producing iron /oxygen complexes such as ferryl and perferryl ions that extract a hydrogen atom from the polyunsaturated fatty acid of membranes [29 /33]. ADM /iron complexes efficiently cause lipid peroxidation [29,30,34]. ICRF-187, a strong iron-chelator that has an EDTA-like structure, inhibits cardiomyopathy induced by ADM and iron [35]. In this study, however, iron-chelators, including EDTA and DETAPAC had no effect on SDH inactivation, indicating that iron does not affect the inactivation of SDH. Even under anaerobic conditions, ADM inactivated SDH in the presence of HRP /H2O2, indicating no participation of oxygen. Oxidized ADM may directly react with enzymes to change the conformation of SDH, leading to loss of enzyme activity of SDH. GSH efficiently blocked SDH inactivation. We previously showed that SH loss of CK was caused by ADM with HRP /H2O2 [9]. Both CK and SDH are typical SH enzymes. Presumably, sensitivity of SDH to ADM with HRP /H2O2 is due to oxidation of the SH group at the active center. ADM-induced free radical generation [5,7], lipid peroxidation [29 /31,34] or mitochondrial enzyme inactivation [35] is at extremely high concentrations of ADM. Non-oxidative damage of mitochondrial electron transport components needs several 100 mM of ADM [11]. Mitochondrial membranes, as major targets of ADM toxicity,

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may accumulate a particularly high ADM concentration because of the affinity of ADM for cardiolipin [36,37]. However, at doses of bolus administration varying between 15 and 90 mg/m2, the maximal initial concentration detected is approximately 5 mM [38,39]. In this study, the IC50 of ADM that inactivated SDH was about 0.8 mM. These findings may reflect the mechanism associated with the clinical use of ADM. Congestive heart failure is caused by anthracycline drugs [12 /14]. Platel et al. [12] compared ADM with idarubicin in rat cardiotoxicity and found that similar general toxicity symptoms were obtained for a dose ratio of 1:4 (idarubicin:ADM). Also, the actions of aclarubicin were weak compared to those of ADM or pirarubicin [14]. Furthermore, Hirano et al. [13] investigated cardiotoxicity of ADM, epirubicin and pirarubicin by using electrocardiography and histopathology and showed that cardiotoxic actions of pirarubicin are weaker than those of ADM and epirubicin. These findings suggest that some cardiotoxicity by anthracyclines is explained by inactivations of mitochondrial SDH and CK through oxidative metabolism of anthracyclines aglycone. Daunorubicin is rapidly oxidized by lactoperoxidase / H2O2 in the presence of nitrite [40]. Mitochondrial SDH and CK were inactivated with loss of red color of anthracyclines in this study. Although data are not shown, we confirmed that semiquinone radical formation was caused through reduction of the C ring in ADM by xanthine oxidase or microsomes under anaerobic conditions [27,28]. The red color of ADM was constant. However, HRP easily oxidizes hydroquinone to the semiquinone [25], strongly suggesting that the hydroquinone structure in the B ring of anthracyclines is oxidized by HRP /H2O2 with loss of red color. Oxidation of anthracyclines should have a crucial role in the inactivation of enzymes. Presumably, oxidative semiquinone or quinone formed from the B ring of anthracyclines participates in the enzyme inactivation. In this study, HRP was used as a model enzyme. The heart muscle of mammals has a large amount of myoglobin. Hydrogen peroxide is produced in various biological reactions. Ferrylmyoglobin, which is produced from myoglobin with H2O2,

acts like peroxidase complex I to be capable of oxidizing a wide range of reducing substrates, such as phenol and aromatic amine [41,42]. ADM, which contain a hydroquinone structure on the B ring, may be a good substrate for ferrylmyoglobin.

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