Effect of iron and ascorbate on cyclosporine-induced oxidative damage of kidney mitochondria and microsomes

Pharmacological Research, Vol. 43, No. 2, 2001 doi:10.1006/phrs.2000.0759, available online at http://www.idealibrary.com on

EFFECT OF IRON AND ASCORBATE ON CYCLOSPORINE-INDUCED OXIDATIVE DAMAGE OF KIDNEY MITOCHONDRIA AND MICROSOMES SUK HA LEE, YOUNG CHUL YOON, YOON YOUNG JANG, JIN HO SONG, EUN SOOK HAN and CHUNG SOO LEE∗ Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul 156-756, South Korea Accepted 7 September 2000

The stimulatory effect of iron and ascorbate on the damaging action of cyclosporine in kidney mitochondria, microsomes and epithelial cells was examined. Cyclosporine induced malondialdehyde formation and hydrogen peroxide production in mitochondria and attenuated the activity of MnSOD and glutathione peroxidase. The damaging effect of cyclosporine (50 µM) plus Fe2+ (20 µM) on mitochondrial and microsomal lipids and proteins as well as mitochondrial thiols was greater than the summation of the oxidizing action of cyclosporine alone and Fe2+ alone. As for tissue components, iron enhanced cyclosporine-induced viability loss in kidney epithelial cells. Fe2+ , EDTA and H2 O2 -induced 2-α deoxyribose degradation was attenuated by 10 mM DMSO and 200 µM DTPA but not affected by 200 µM cyclosporine. The addition of Fe2+ caused a change in the absorbance spectrum of cyclosporine in the wavelength range 230–350 nm. The simultaneous addition of cyclosporine (50 µM) and ascorbate (100 µM) showed the enhanced peroxidative effect on mitochondrial and microsomal lipids, which was inhibited by DTPA and EDTA (1 mM). Similar to iron, ascorbate enhanced cyclosporine-induced cell viability loss. The results show that iron and ascorbate promote the damaging action of cyclosporine in kidney cortex mitochondria and microsomes and in kidney epithelial cells, which may contribute to the c 2001 Academic Press enhancement of cyclosporine-induced nephrotoxicity.

K EY WORDS : kidney damage, cyclosporine, iron, ascorbate, promoting effect.

INTRODUCTION Cyclosporine has been used in the prevention and treatment of organ transplant rejection and in the treatment of autoimmune diseases [1]. It has a selective inhibitory effect on T-lymphocytes, suppressing the early response to antigenic and regulatory stimuli. The clinical uses of the drug are restricted by side effects; such as nephrotoxicity, hepatotoxicity and neurotoxicity. The major problems associated with cyclosporine treatment occur in the kidney and liver. The underlying mechanisms leading to nephrotoxicity have not been properly investigated. A number of mediators and mechanisms have been proposed as the cause of cyclosporine-induced nephrotoxicity [2]. The causative factors appear to include renal vasoconstriction, metabolism of the drug, thiol oxidation and formation of free radicals. The role of free radicals has been postulated in the pathogenic mechanism of the cytotoxicity of cy∗ Corresponding author. Department of Pharmacology, College of

Medicine, Chung-Ang University, 221 Heuk-Suk Dong, Dong-Jak Gu, Seoul 156-756, South Korea. E-mail: [email protected] 1043–6618/01/020161–11/$35.00/0

closporine [3, 4]. Potential sources of free radicals in the kidney and liver include NADPH-oxidase mediated formation of oxidants in mesangial cells, uncoupling of the cytochrome P450 monooxygenase system and the mitochondrial respiratory chain [2, 5]. Reactive oxygen species are known to be produced during metabolism of cyclosporine. In rat-cultured hepatocytes, cyclosporine increased reactive oxygen species production and the formation of thiobarbituric acid (TBA) reactive substances and induced loss of protein thiols. Administration of cyclosporine into renal transplant patients [6] and animals [4] caused alteration of endogenous antioxidant enzyme activities. Antioxidants, including vitamin E, are found to inhibit hydrogen peroxide production induced by cyclosporine in cultured human mesangial cells [7], to reduce lipid peroxidation of renal microsomes [8], to prevent change of antioxidant enzyme activity in the kidney tissues and to restore a decrease in renal function. Cyclosporine administration to vitamin E and selenium deficient rats enhanced production of TBA reactive substances and reduced renal function. Glutathione of reduced form plays an important role in c 2001 Academic Press

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the detoxication of xenobiotics and in the protection of cell components against oxidative damage. A decrease of glutathione concentration in the renal cortex is a common biochemical response to many nephrotoxic agents [9, 10]. Cyclosporine significantly decreased the ratio of GSH/GSSG within the renal cortex. The glutathione disulfide-reducing agent dithiothreitol is found to attenuate the decrease of the GSH/GSSG redox state caused by cyclosporine and to inhibit the cyclosporine toxicity [11]. The redox state of cellular glutathione significantly affected the cyclosporine toxicity. Metal ions significantly catalyze the formation of highly reactive oxygen species and effectively promote oxidative tissue damage [12, 13]. The kidney, particularly the proximal tubule, is rich in ferritin [14]. Other sources of iron within the kidney are haem proteins which include mitochondrial cytochromes, cytochrome P450, and catalase. Extrarenal haem proteins, such as haemoglobin and myoglobin, might also serve as sources of iron [15]. The oxidoreduction of iron is considered to play a central role in lipid peroxidation. The autoxidation of iron produces reactive oxygen metabolites, such as hydroxyl radicals, the perferryl ion and the ferryl ion, and these oxygen metabolites appear to be involved in the initiation of lipid peroxidation [12]. Erythrocytosis is found to be prevalent in cyclosporinetreated renal transplant patients [16]. Nonenzymatic ascorbate-induced lipid peroxidation has been demonstrated to increase in cyclosporin A-administered rats [17]. The iron chelator desferrioxamine inhibited cyclosporine-induced contraction of renal cortex glomeruli, which suggested the implication of hydroxyl radicals and lipid peroxidation in glomerular contraction [18]. Cytochrome P450 inhibitors are found to attenuate cyclosporine-induced microsomal lipid peroxidation [19]. Therefore, the implication of iron in cyclosporine-induced nephrotoxicity is suggested. However, the promoting effect of metal ions, including iron, on the cytotoxic action of cyclosporine and the interaction of iron and cyclosporine has not been elucidated. Ascorbate has been shown to promote or attenuate oxidative damage depending on the concentration [20]. Low concentration of ascorbate appears to promote metalion-caused reactive oxygen species formation and shows a pro-oxidant effect. Like iron, the role of ascorbate in cyclosporine cytotoxicity is not well understood. The present study explored the role of iron and ascorbate in the cytotoxic action of cyclosporine on the kidney. We examined their effect on antioxidant enzyme activities, the formation of peroxidation products and thiol oxidation and reactive oxygen species formation in kidney cortex mitochondria and microsomes and on the viability of kidney epithelial cells. The results show that iron and ascorbate promote the damaging action of cyclosporine on kidney cortex mitochondria and microsomes and on kidney epithelial cells, which may contribute to enhancement of cyclosporineinduced nephrotoxicity.

Pharmacological Research, Vol. 43, No. 2, 2001

MATERIALS AND METHODS

Materials Cyclosporine (Sandimmun Neoral) was obtained from Sandoz Pharmaceutical Ltd., Switzerland. Superoxide dismutase (from bovine Blood, SOD), catalase (from bovine liver), dimethyl sulfoxide (DMSO), 1,4dizabicyclo (2,2,2) octane (DABCO), diethylenetriamine pentaacetic acid (DTPA), ethylenediamine-tetraacetic acid (EDTA), ascorbic acid, ferricytochrome c, xanthine, xanthine oxidase, glutathione (reduced form), glutathione reductase, NADPH, t-butyl hydroperoxide, phenol red, horseradish peroxidase, 2-thiobarbituric acid (TBA), 2,4-dinitrophenylhydrazine, guanidine, 5,50 dithiobis-(2-nitrobenzoic acid), 3-(4,5-dimethylthiazoyl2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and 2-α deoxyribose were purchased from Sigma-Aldrich Inc. Fetal bovine serum was obtained from Gibco-BRL. Other chemicals used were of analytical grade. In the Figures and Tables, cyclosporine is denoted as CsA.

Isolation of rat kidney cortex mitochondria and microsomes Mitochondria were prepared from the kidney cortex of male Sprague–Dawley rats weighing about 200 g [21]. The kidney was removed and sliced in a medium labeled I (250 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM EDTA, 120 mM KCl and 50 mM Tris-HCl, pH 7.4). The tissue was homogenized with a polytron homogenizer (Brinkman Instruments, Ont., Canada, model PT-20). The homogenate was centrifuged at 755 g for 5 min, and the resulting supernatant was centrifuged at 13 300 g for 15 min. The pellet was suspended in a medium labeled II (250 mM mannitol, 70 mM sucrose, 10 mM HEPES and 50 mM Tris-HCl, pH 7.4 or 120 mM KCl and 50 mM Tris-HCl, pH 7.4) and washed twice with the same buffer by centrifugation at 13 300 g for 15 min. The final mitochondrial pellet was suspended in medium II. The protein concentration was determined by the Bradford method using the Bio-Rad protein assay kit. The supernatant, obtained from the first 13 300 g centrifugation, was centrifuged at 100 000 g for 60 min. The resulting pellet was suspended in a medium (120 mM KCl and 50 mM Tris-HCl, pH 7.4), and the pellet was washed with the same buffer by centrifugation at 20 000 g for 30 min. The suspension was recentrifuged at 100 000 g for 60 min, and the final microsomal pellet was suspended in the KCl-Tris medium [22].

Cell culture The kidney epithelial cell line (TCMK-1, mouse) was obtained from the Korean cell line bank. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units ml−1 penicillin and 100 µg ml−1 streptomycin as described in the manual of the cell line bank. Cells were subcultured every 5–7 days.

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Measurement of MnSOD activity Mitochondria (0.1 mg protein ml−1 ) were suspended in reaction mixtures consisting of 10 µM ferricytochrome c, 0.03% sodium deoxycholate, 100 µM EDTA, 2 mM KCN, 50 µM xanthine and 50 mM KH2 PO4 , pH 7.4. The reaction was started by addition of xanthine oxidase, and absorbance was measured at 550 nm. The absorbance change of cytochrome-c induced by the enzyme without mitochondria was adjusted to show 0.021–0.025 at the same wavelength. Fifty percent inhibition of cytochromec reduction is defined as 1 unit of enzyme activity [23].

Measurement of glutathione peroxidase activity Glutathione peroxidase activity was assayed by the method given in [24]. Mitochondria (0.1 mg protein ml−1 ) were suspended in 25 ◦ C reaction mixtures containing 1 mM GSH, 0.96 U ml−1 glutathione reductase, 100 µM EDTA and 50 mM KH2 PO4 , pH 7.4. The reaction was started by addition of 150 µM NADPH and 120 mM t-butyl hydroperoxide, sequentially, and then absorbance was measured at 340 nm. The units of enzyme activity were calculated using an extinction coefficient of 6.22 × 103 M−1 cm−1 . One unit of the enzyme is defined as 1 nM NADPH consumed per minute.

Measurement of hydrogen peroxide production ml−1 )

Mitochondrial suspensions (1 mg protein were incubated in 1 ml of potassium phosphate buffer containing cyclosporine, 0.1 mg ml−1 phenol red, 0.2 mg ml−1 horseradish peroxidase, 120 mM KCl and 0.1 mM sodium azide for 1 h at 30 ◦ C. The reaction was terminated by adding 100 µl of 1 N NaOH, and mixtures were centrifuged at 13 000 g for 8 min. The absorbance of supernatant was measured at 610 nm, and the concentration of hydrogen peroxide produced was calculated using hydrogen peroxide solution as standard [25].

Measurement of lipid peroxidation Lipid peroxidation of mitochondria and microsomes was estimated by measuring malondialdehyde (MDA) concentration by the thiobarbituric acid method. Mitochondria and microsomes (0.4 mg protein ml−1 ) were suspended in 1 ml of reaction mixture consisting of 150 mM KCl, 0.1 mM sodium azide and 50 mM NaH2 PO4 , pH 7.4. The reaction was started by addition of cyclosporine and continued for 30 min. The reaction was terminated by adding 1 ml of 1% TBA in 50 mM NaOH and 1 ml of 2.8% trichloroacetic acid. The absorbance of chromophore developed was measured at 532 nm. The concentration of MDA was expressed as nmol mg−1 protein using the molar extinction coefficient of 1.56 × 105 M−1 cm−1 [26, 27].

which contain 1 mg protein ml−1 of mitochondria or microsomes, were treated with cyclosporine and FeSO4 (or ascorbate) for 30 min. A mixture of 4 ml of 10 mM 2,4-dinitrophenylhydrazine in 2.5 M HCl was added to the mixtures, and the tubes were left for 1 h at room temperature in the dark. A 5 ml volume of 20% TCA solution was added, and the tubes were placed in an ice bucket for 10 min. After centrifugation, pellets were washed three times with 4 ml of ethanol : ethyl acetate (1 : 1) solution. The final pellets were dissolved in 2 ml of 6 M guanidine HCl solution and left for 15 min at 37 ◦ C with mixing. Absorbance of the supernatants was measured at 370 nm, and protein carbonyls were calculated using the molar extinction coefficient of 2.2 × 104 M−1 cm−1 .

Measurement of mitochondrial thiol content Kidney mitochondria (1 mg protein ml−1 ) suspended in 100 mM Tris-HCl, pH 7.4 buffer medium were treated with 50 µM cyclosporine (or with 20 µM iron) for the indicated times at 37 ◦ C. The thiol content in mitochondria was determined using 5,50 -dithiobis-(2nitrobenzoic acid) as described in the method of [29].

MTT assay Cell viability was measured by using the MTT assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases. Cells were plated at a density of 2 × 105 cells per 200 µl medium in a 96-well plate and treated with 50 µM cyclosporine in the presence of iron or ascorbate for 30 min. The medium was incubated with 5 µl of 10 mg ml−1 MTT solution for 1 h at 37 ◦ C. The culture medium was removed, and 100 µl of DMSO was added to each well to dissolve formazan. The absorbance was measured at 570 nm in a microplate reader (Molecular Devices Co., Spectra MAX 340, Sunnyvale, CA, U.S.A.). Cell viability loss was expressed as a percentage of the control culture [30, 31].

Measurement of 2-α deoxyribose degradation Hydroxyl radical production was estimated by the assay of MDA chromogen formation due to 2-α deoxyribose degradation [32]. The reaction mixtures contained 2 mM 2-α deoxyribose, cyclosporine, 50 µM FeSO4 , 50 µM EDTA, 500 µM H2 O2 , 150 mM KCl and 50 mM NaH2 PO4 buffer, pH 7.4 in a final volume of 1 ml. After 30 min of incubation, the reaction was terminated by adding 1 ml of 1% TBA in 50 mM NaOH and 1 ml of 2.8% trichloroacetic acid. The reaction mixtures were heated to promote chromophore development, and absorbance was measured at 532 nm.

Wavelength scan of cyclosporine Measurement of carbonyl groups Protein oxidation in mitochondria and microsomes was determined by carbonyl assay using 2,4dinitrophenylhydrazine [28]. The 1 ml reaction mixtures,

Cyclosporine (50 µM) was treated with the stated concentration of iron (or copper, ascorbate and NADPH) for 30 min, and then absorbance change was measured at variable wavelengths between 230 and 350 nm.

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MnSOD and GSH peroxidase activity

MDA and H2O2 production (nmol per mg of protein)

164

MDA H2O2

2

1

40

30

20

10

0 0 0 10

50 CsA(µM)

100

Fig. 1. Cyclosporine induced lipid peroxidation and hydrogen peroxide production in kidney mitochondria. Kidney cortex mitochondria (0.4 mg protein ml−1 for MDA assay and 1 mg protein ml−1 for hydrogen peroxide production assay) were treated with varying concentrations of cyclosporine for 30 min. The MDA and hydrogen peroxide formed were assayed as described in the Materials and Methods section. Data are mean ± SEM, n = 4. ∗ P < 0.05, ∗∗ P < 0.01; significantly different from no addition of cyclosporine.

Statistical analysis The statistical analysis on the data obtained from various experiments was performed using the Mann– Whitney test. Data were expressed as mean ± SEM. The results under ∗ P < 0.05 are regarded as having statistical significance.

RESULTS

The effect of cyclosporine on kidney mitochondrial antioxidant enzymes The involvement of reactive oxygen species in cyclosporine cytotoxicity in the kidney was studied with activity changes of endogenous antioxidant enzymes and oxidant hydrogen peroxide production. When mitochondria of the kidney cortex were incubated with cyclosporine, MDA formation was increased with increasing drug concentrations (Fig. 1). Cyclosporine (50 µM) produced 1.98 ± 0.07 nmol mg−1 per protein of MDA, n = 4 for 30 min incubation. Since reactive oxygen species are thought to be produced during metabolism of cyclosporine [3], the cyclosporine-induced oxidant production in reaction mixtures containing kidney mitochondria was examined with hydrogen peroxide production. As shown in Fig. 1, hydrogen peroxide production by mitochondria was enhanced with increasing concentrations of cyclosporine. Incubation of mitochondria with 50 µM cyclosporine produced 2.08 ± 0.23 nmol of H2 O2 per mg of protein per 30 min, n = 4. The effect of cyclosporine on the activity of antioxi-

MnSOD GSH peroxidase

0 10

50 CsA(µM)

100

Fig. 2. Cyclosporine induced inhibition of MnSOD and glutathione peroxidase activities. Kidney cortex mitochondria were treated with varying concentrations of cyclosporine for 30 min. Enzyme activities were assayed as described in the Materials and Methods section. Data are mean ± SEM, n = 4. ∗∗ P < 0.01; significantly different from no addition of cyclosporine.

dant enzymes in kidney mitochondria, which may reflect oxidative stress, was elucidated. Fig. 2 shows that cyclosporine attenuated the activities of MnSOD and glutathione peroxidase in a dose-dependent manner, and at 50 µM concentration, the drug revealed 37 and 35% of inhibition, respectively.

The effect of iron on cyclosporine-induced oxidative damage of kidney mitochondria and microsomes and kidney epithelial cells Cyclosporine cytotoxicity was evaluated with the peroxidation of kidney cortex mitochondrial and microsomal components. The concentration of cyclosporine used in the present study is based on previously reported data [3, 5, 19]. Iron, which is presented in µM concentrations within the tissues, is well known to promote tissue damage caused by reactive oxygen species [12]. In this respect, the effect of iron on cyclosporine-induced oxidative damage of the kidney was examined. Figure 3 shows the enhanced damaging effect of cyclosporine in the presence of iron (FeSO4 ) on the kidney tissue components. Cyclosporine (50 µM) and iron (20 µM) caused oxidative damage of mitochondria and microsomes. The effect of the simultaneously added cyclosporine and iron on the peroxidation of lipids and proteins in mitochondria and microsomes was elucidated. The damaging effect of cyclosporine plus iron on mitochondrial and microsomal lipids and proteins was greater than the summation of the oxidizing action of cyclosporine alone and iron alone. They showed promoting effects on MDA formation in mitochondria and microsomes amounting to 65 and 36%, respectively, and on carbonyl formation via protein oxidation by 29 and 12%. Unlike iron, 20 µM Cu2+ did not reveal the stimulatory effect on 50 µM cyclosporine-induced mitochondrial lipid peroxidation.

Carbonyls (nmol per mg of protein)

0

ne OD lase SO CO S ata M AB D D C

No

M

Fig. 3. Damaging action of cyclosporine and iron(II) on mitochondria and microsomes. Kidney mitochondria and microsomes were treated with 50 µM cyclosporine and 20 µM FeSO4 (or CuSO4 ). Data are mean ± SEM, n = 4. ∗ P < 0.05, ∗∗ P < 0.01; significantly different from the summation of cyclosporine alone plus Fe2+ alone.

The reactive oxygen species, which may be involved in the cyclosporine plus iron-induced peroxidation of kidney mitochondrial lipids and proteins, were examined. Concentrations of oxidant scavengers used in this study are based on the reported data [19, 33, 34], and the compounds alone at given concentrations did not show significant damaging effects on mitochondria (data not shown). As shown in Fig. 4, SOD (30 µg ml−1 ), a scavenger of the superoxide anion, 30 µg ml−1 catalase (a scavenger of hydrogen peroxide) and 10 mM DMSO, a scavenger of hydroxyl radicals, attenuated cyclosporine plus ironinduced MDA formation, whereas the protective effect of 10 mM DABCO, a quencher of singlet oxygen, was not detected. The indicated concentrations of oxidant scavengers exhibited different protective effects on the cyclosporine plus iron-induced peroxidation of mitochondrial proteins. In this experiment, dissimilar to lipid peroxidation, DMSO and DABCO showed protective effects on mitochondrial protein oxidation greater than lipid peroxidation, whereas SOD did not exhibit any interfering effect. The effect of iron on cyclosporine cytotoxicity was examined using kidney epithelial cells. When kidney epithelial cells were treated with 50 µM cyclosporine for 30 min, cell viability reduced by 19% in the MTT assay. Similar to kidney mitochondria and microsomes, 20 µM iron enhanced cyclosporine-induced viability loss in kidney epithelial cells by 34% (Fig. 5). Like iron, the ascorbate (100 µM) stimulated cyclosporine toxicity on kidney epithelial cells. Under the same experimental condition, iron and ascorbate alone caused cell viability loss of 7 and 5%, respectively.

Interaction of cyclosporine with iron-induced oxidative stress To ascertain whether the peroxidative effect of cyclosporine plus iron on kidney mitochondrial and

2

1 0 ne OD lase SO CO S ata M AB D D C

No

Fig. 4. The effect of oxidant scavengers on cyclosporine and ironinduced mitochondrial damage. Kidney mitochondria (0.4 mg protein ml−1 ) were treated with 50 µM cyclosporine and 20 µM FeSO4 in the presence of oxidant scavengers. SOD, 30 µg ml−1 ; catalase, 30 µg ml−1 ; DMSO, 10 mM and DABCO, 10 mM. Data are mean ± SEM, n = 4. ∗ P < 0.05, ∗∗ P < 0.01; significantly different from no addition of cyclosporine.

40 35 30 25 20 15 10 5 0

Cs As A co + A rba sc te or ba te

M

ito

icr

es

om os

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A

M

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nd

o ch

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Cs

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% Cell viability loss

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MDA (nmol per mg of protein)

CsA Fe2+ CsA + Fe2+

Cu2+

MDA (nmol per mg of protein)

12

Carbonyls (nmol per mg of protein)

Pharmacological Research, Vol. 43, No. 2, 2001

Fig. 5. The effect of iron and ascorbate on cyclosporine cytotoxicity. Kidney epithelial cells (2 × 105 cells per 200 µl) were treated with 50 µM cyclosporine in the presence of 20 µM FeSO4 (or 100 µM ascorbate) for 30 min. Data are mean ± SEM of cell viability loss, n = 5. ∗∗ P < 0.01; significantly different from the summation of cyclosporine alone plus iron (or ascorbate) alone.

microsomal components was greater than the summation of that of cyclosporine alone and iron alone, the present study examined a possible interaction of cyclosporine with iron in the tissue damage. Figure 6 shows that in the presence of a fixed concentration (10 µM) of iron, cyclosporine stimulated peroxidation of mitochondrial lipids in a dose-dependent manner, and at 100 µM concentration, 25% stimulation was shown. In the present study, 5 and 10 µM of cyclosporine did not show a significant enhancing effect. In a similar experiment, the

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effect of a fixed concentration (50 µM) of cyclosporine on mitochondrial lipid peroxidation was enhanced with increasing concentrations of iron. Metal ion chelators are known to enhance iron-induced peroxidation of unsaturated fatty acids [35, 36]. Gentamicin is found to exhibit oxidative damage through the formation of an iron–gentamicin complex and to inhibit the oxidation of salicylate caused by an iron–EDTA complex [37]. In these respects, the effect of cyclosporine on the iron–EDTA complex-induced 2-α deoxyribose degradation, which is used as the sensitive method for hydroxyl radical detection in a similar fashion to the salicylate hydroxylation assay, was examined. Fe2+ (50 µM), 50 µM EDTA and 500 µM H2 O2 showed 2-α deoxyribose degradation, which was attenuated by addition of 10 mM DMSO. DTPA (200 µM), a specific iron chelator, decreased the peroxidative action of Fe2+ , EDTA and H2 O2 on 2-α deoxyribose. In contrast to DTPA, the addition of cyclosporine (200 µM) showed little or no stimulatory effect on Fe2+ , EDTA and H2 O2 induced deoxyribose degradation, as shown in Fig. 7. The interaction of cyclosporine with iron was elucidated through the change of the absorbance spectrum of cyclosporine. In Fig. 8, the addition of 20 µM Fe2+ caused a change in the absorbance spectrum of cyclosporine (50 µM) in the wavelength range between 230 and 350 nm. In contrast, this finding was not seen in cyclosporine treated with the same concentration of Cu2+ .

Effect of ascorbate on cyclosporine-induced oxidative kidney damage The cellular physiological concentrations of ascorbate are found to be in the range of µM to mM. Low con-

+

+

DM

Fig. 7. The effect of cyclosporine on iron, EDTA and hydrogen peroxide (IEH)-induced deoxyribose degradation. Cyclosporine (200 µM) was added to reaction mixtures containing 50 µM FeSO4 , 50 µM EDTA, 500 µM H2 O2 and 2 mM 2-α deoxyribose. DMSO, 10 mM and DTPA, 200 µM. Data are mean ± SEM, n = 4. ∗∗ P < 0.01; significantly different from IEH.

1.0 0.8

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◦

•

CsA 0.4 0.2 Fe2+ 0.0 230

254

278

302

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350

302

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350

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Fig. 6. The effect of iron on mitochondrial lipid peroxidation caused by cyclosporine. Kidney mitochondria were treated with either various concentrations of cyclosporine in the presence of 10 µM FeSO4 ( ) or with 50 µM cyclosporine plus various concentrations of iron ( ). Data are mean ± SEM, n = 5. Data represent the percentage stimulation of cyclosporine and iron summation from the addition of cyclosporine alone and iron alone.

SO

PA

DT

CsA 0.4 0.2 Cu2+ 0.0 230

254

278

Wavelength Fig. 8. Change of absorbance spectrum of cyclosporine induced by iron. Cyclosporine (100 µM) was treated with 50 µM FeSO4 , (or CuSO4 ) for 10 min. Absorbance was measured at variable wavelengths between 230 and 350 nm.

Pharmacological Research, Vol. 43, No. 2, 2001

Effect of iron on cyclosporine-induced thiol oxidation Thiol groups are thought to play an important role in the maintenance of cell functions and cell membrane integrity [10, 38]. Thiol groups in kidney mitochondria were oxidized by addition of 50 µM cyclosporine. The thiol oxidation in mitochondria was proceeded with increasing incubation times, and at 30 min of incubation, 21% of the thiol groups were oxidized (Fig. 10). As shown in Fig. 11, the simultaneous addition of 50 µM cyclosporine and 20 µM iron exerted an enhanced effect on the oxidation of mitochondrial thiols for up to 15 min incubation, whereas their promoting effect was not observed at 30 min incubation time.

DISCUSSION The reactive oxygen species-induced cytotoxicity is promoted by metal ions, particularly iron. Oxidoreduction of metal ions is thought to play a central role in tissue destruction, and iron effectively catalyzes the tissue damage by oxidants and free radicals. Autoxidation of iron produces reactive oxygen metabolites, which appear to cause damage to biological molecules. Gentamicin has been shown to enhance the production of reactive oxygen species [37, 39], and its action is promoted by iron [2]. In both Epstein–Barr virus-transformed lymphoblastoid cells and in cell-free systems, gentamicin-enhanced free radical production was suppressed by iron chelators [40]. Inhibitors of the iron source cytochrome P450, are found to prevent hypoxic renal cell injury [41]. Desferrioxamine attenuated the contraction of renal cortex glomeruli induced by cyclosporine. These studies indicate the im-

1.0 0.8 CsA + Ascorbate Absorbance

0.6 CsA 0.4 0.2 Ascorbate 0.0 230

254

278

302

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1.0 0.8

CsA + NADPH

0.6 Absorbance

centrations of ascorbate appear to stimulate oxidative tissue damage [20]. Similar to the effect of cyclosporine and iron, the simultaneous addition of cyclosporine (50 µM) and ascorbate (100 µM) showed a promoting action on MDA formation in kidney mitochondria and microsomes, and they exhibited 140 and 66% enhancement, respectively. The stimulatory effect of cyclosporine and ascorbate was also found in kidney epithelial cells (Fig. 5). However, the enhancing effect of cyclosporine and ascorbate on carbonyl formation in kidney mitochondria and microsomes was not apparent (Table I). The implication of iron in the cyclosporine plus ascorbate-induced damage of mitochondrial lipids and proteins was explored. Addition of 1 mM of DTPA and EDTA significantly attenuated the cyclosporine and ascorbate-caused mitochondrial oxidative damage. The interaction of cyclosporine with ascorbate was examined through the change in the absorbance spectrum of cyclosporine as shown in Fig. 9. When 50 µM of cyclosporine was treated with 100 µM ascorbate, the absorbance spectrum of cyclosporine changed. This finding was similar to the absorbance spectrum change observed when cyclosporine is mixed with NADPH.

167

CsA 0.4 NADPH

0.2 0.0 230

254

278

302

326

350

Wavelength Fig. 9. Change of absorbance spectrum of cyclosporine induced by ascorbate. Cyclosporine (100 µM) was treated with 100 µM ascorbate (or NADPH) for 10 min. Absorbance was measured at variable wavelengths between 230 and 350 nm.

plication of iron in cyclosporine-induced tissue damage. It has been shown that iron is released from mitochondrial cytochrome P450 during hypoxia/reperfusion in the kidney [41] and from mitochondria exposed to the nephrotoxin gentamicin [42]. Cytochrome P450 is suggested as the critical source of iron. Cytochrome P450 inhibitors are found to attenuate hydroxyl radical production induced by reoxygenation in the kidney and to inhibit cyclosporine-caused peroxidation of liver microsomes. In contrast to the findings indicate that the catalyzing action of iron in gentamicin-induced nephrotoxicity and in reperfusion-evoked renal injury, the role of iron in cyclosporine cytotoxicity has not been clearly elucidated. Cyclosporine induced oxidative damage of lipids and proteins in the kidney cortex mitochondria and microsomes. In the reaction mixtures containing mitochondria, cyclosporine produced MDA formation, stimulated hydrogen peroxide production and depressed the activities of endogenous antioxidant enzymes, MnSOD and glutathione peroxidase. Decreased antioxidant enzyme activities may reflect the role of oxidative stress in the cyclosporine toxicity on kidney mitochondria. With hy-

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Pharmacological Research, Vol. 43, No. 2, 2001 Table I Damaging action of cyclosporine and ascorbate on kidney mitochondria and microsomes MDA (nmol per mg of protein) Mitochondria Microsomes

CsA Ascorbate CsA + Ascorbate CsA + Ascorbate + DTPA 1 mM CsA + Ascorbate + EDTA 1 mM

2.02 ± 0.06 0.39 ± 0.01 ß5.76 ± 0.09∗∗

Carbonyls (nmol per mg of protein) Mitochondria Microsomes

3.26 ± 0.11 0.97 ± 0.08 ß7.01 ± 0.10∗∗

1.03 ± 0.07 1.04 ± 0.05 2.10 ± 0.10

2.14 ± 0.10 0.64 ± 0.03 2.48 ± 0.12

1.05 ± 0.06††

—

1.07 ± 0.08††

—

0.88 ± 0.03††

—

0.93 ± 0.06††

—-

36 Thiol oxidation (nmol per mg of protein)

Thiol oxidation (nmol per mg of protein)

Kidney mitochondria and microsomes (0.4 mg protein ml−1 ) were treated with 50 µM cyclosporine and 100 µM ascorbate. Data are mean ± SEM, n = 4. ∗∗ P < 0.01; significantly different from the summation of cyclosporine alone plus ascorbate alone. †† P < 0.01; significantly different from the summation of cyclosporine plus ascorbate.

34 32 30 28 26 24 0 0

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15 min

30 min

Fig. 10. Cyclosporine induced mitochondrial thiol oxidation. Kidney mitochondria (1 mg protein per ml) were treated with 50 µM cyclosporine. Data are mean ± SEM, n = 4. ∗∗ P < 0.01; significantly different from intact mitochondria.

Fig. 11. The effect of cyclosporine and iron on mitochondrial thiol oxidation. Kidney mitochondria (1 mg protein per ml) were treated with 50 µM cyclosporine and 20 µM FeSO4 for the indicated times. Data are mean ± SEM, n = 5. ∗∗ P < 0.01; significantly different from the summation of cyclosporine alone plus Fe2+ alone.

drogen peroxide formation, cyclosporine-induced MDA formation and the inhibitory effect of reactive oxygen species scavengers on the formation of peroxidative products indicate the involvement of reactive oxygen species in the cyclosporine and iron-induced damage of kidney tissue components. The sensitivity of kidney tissue components to reactive oxygen species, which may be produced from cyclosporine metabolism, was examined. The cyclosporine and iron-induced MDA formation in mitochondria was little or not affected by DMSO and DABCO unlikely to mitochondrial proteins. It has been shown that tissue lipids and proteins, affected by oxidative attack, respond differently to oxidant scavengers [12, 34]. The result indicates that the causative reactive oxygen species chiefly involved in the cyclosporine and iron-induced protein oxidation in kidney mitochondria may be different from lipid peroxidation. Peroxidation of cellular membrane lipids is associated with alteration of membrane function and inactivation of integral enzymes [12, 20]. A trace amount of iron acts as

an effective catalyst for the peroxidation of unsaturated fatty acids. At the basis of physiological concentration, the effect of iron on cyclosporine-induced cytotoxic action was explored. Cyclosporine in the presence of iron showed an enhanced peroxidative action on lipid and protein components of mitochondria and microsomes. This finding was also detected in kidney epithelial cells. The results suggest that cyclosporine and iron exert a damaging synergistic effect on kidney tissues. The interaction of cyclosporine with iron in the cytotoxic action may be supported by the result which shows that in the presence of fixed concentration of iron, lipid peroxidation was enhanced with increasing concentrations of cyclosporine. Similar to this finding, their enhancing effect on mitochondrial damage was seen in the fixed amount of cyclosporine. The iron oxygen complex is considered to be the causative agent of lipid peroxidation rather than the highly reactive oxygen species hydroxyl radicals [12]. In agreement with this review, the well known scavenger of hydroxyl radicals, DMSO,

Pharmacological Research, Vol. 43, No. 2, 2001

showed little inhibitory effect on mitochondrial lipid peroxidation induced by cyclosporine and iron. Gentamicin is suggested as an iron chelator, and the catalytic action of the iron–gentamicin complex in free radical formation in kidney tissue is postulated [37]. The cyclosporine– iron interaction may also be confirmed by the small inhibitory effect of DMSO on the peroxidative action of cyclosporine and iron on mitochondrial lipids and by the absorbance spectrum change of cyclosporine treated with iron, which was not seen for the addition of copper. Gentamicin has been shown to inhibit the oxidation of salicylate induced by the iron–EDTA complex, which is an effective producer of hydroxyl radicals. In order to examine if cyclosporine has a chelating or complex forming action on iron, an experiment similar to salicylate hydroxylation was performed by measuring degradation of 2-α deoxyribose, which is known as a sensitive method for detecting hydroxyl radical production [32, 43]. Implication of hydroxyl radicals in deoxyribose degradation caused by Fe2+ , EDTA and H2 O2 could be confirmed by the depressive effect of DMSO. The iron chelator DTPA significantly attenuated the Fe2+ , EDTA and H2 O2 -induced deoxyribose degradation. However, under the same experimental conditions, cyclosporine did not show any significant (inhibitory or stimulatory) effect on the iron-mediated deoxyribose degradation. Therefore, the chemical property of the cyclosporine– iron interaction product appears to be different from that of the gentamicin–iron complex and that of the metal ion chelator–iron complex. When the ratio of Fe2+ to EDTA or DTPA is 2 : 1, chelated iron is known to exhibit peroxidative action on microsomal lipids [35, 36, 44]. Beyond this ratio, the chelated metal ions do not show the peroxidative action. In contrast to these reports, the same ratio of Fe2+ to cyclosporine did not show the lipid peroxidation stimulatory effect, and regardless of the ratio, their peroxidative effect was enhanced with increasing concentrations of cyclosporine. The finding also supports the above suggestion. The reducing agents, including ascorbate and NADPH, interact with metal ions to promote the formation of reactive oxygen species and stimulate iron-mediated oxidative tissue damage [20]. Ascorbate may act as antioxidant at lower concentrations, but as a pro-oxidant at higher concentrations [45, 46]. This active compound is presented at a millimolar concentration at the kidney [20]. Ascorbate administration shows an improvement in the activities of antioxidant enzymes, such as SOD and glutathione peroxidase, and partially attenuates the subcellular damage under oxidative stress. The cytotoxic action of cyclosporine on cultured rat hepatocytes was attenuated by addition of ascorbate [5]. In contrast, the ascorbate-induced lipid peroxidation of the kidney and liver in rats is found to be enhanced by cyclosporine treatment [17]. It has been shown that ascorbate (1 mM) causes apoptosis in rat pheochromocytoma PC12 cells, and a 0.1 mM concentration potentiates dopamine-induced apoptosis [47]. Similar

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to this finding, ascorbate is demonstrated to act as a pro-oxidant for the induction of apoptosis in human promyelocytic leukemic HL-60 cells [48] and to potentiate dopamine-induced apoptosis in a human neuronal cell line [49]. Ascorbate promoted the damaging effect of cyclosporine on mitochondrial and microsomal lipids and on kidney epithelial cells in agreement with previous reports on the pro-oxidant action of ascorbate, while the enhancing effect was not seen in carbonyl formation in the same tissues as protein oxidation. Therefore, the interaction of cyclosporine with ascorbate may exhibit a different damaging effect on kidney mitochondria, and mitochondrial proteins compared with the same tissue lipids. The pro-oxidant action of ascorbate is known to be mediated by the recycling of iron from the ferric to ferrous state [50]. Involvement of iron in the damaging action of cyclosporine plus ascorbate on mitochondrial lipids and proteins could be postulated by the depressant effect of iron and metal chelators, DTPA and EDTA, on the formation of MDA and carbonyls. Cyclosporine mixed with ascorbate showed an absorbance spectrum change similar to that found in NADPH-treated cyclosporine. However, in contrast to ascorbate, our report shows that the reducing agents, such as NADPH, NADH and GSH, do not stimulate the peroxidative action of cyclosporine in kidney mitochondrial and microsomal lipids and rather inhibited it [51]. This finding suggests the interaction of cyclosporine with ascorbate in the cyclosporine-induced tissue damage. Protein thiols are considered to play an important role in the maintenance of membrane integrity and function. Mitochondrial function has been shown to be affected by the redox state of thiols. A decrease in reduced glutathione may be associated with the formation of membrane permeability transition in mitochondria [38] and with suppression of the mitochondrial electron transport chain [52]. Cyclosporine inhibited glutathione S-transferase, which is probably associated with the kidney toxicity [53]. The weakening or enforcement of the cellular glutathione state is found to increase or decrease cyclosporine cytotoxicity [5]. Cyclosporine directly interacted with mitochondria to produce thiol oxidation. Cyclosporine-induced thiol oxidation in kidney mitochondria may contribute to the nephrotoxic effect. When the kidney mitochondria were treated with cyclosporine and iron, the enhanced damaging effect on thiol oxidation was observed likely to MDA and carbonyl formation up to 15 min of incubation, while the stimulatory effect disappeared with further incubation. Reduced thiols exposed to cyclosporine and iron appear to be oxidized more quickly than tissue lipids and proteins. The results indicate that the significant thiol oxidation at the early phase may contribute to the cytotoxicity of cyclosporine and iron. In conclusion, iron and ascorbate promote the damaging action of cyclosporine on kidney cortex

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mitochondrial and microsomes and on kidney epithelial cells, which may contribute to the enhancement of cyclosporine-induced nephrotoxicity.

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