Induction of Apoptosis by the O-Hydroxyethyl-D(Ser)- cyclosporine A Derivative SDZ IMM 125 in Rat Hepatocytes

The immunosuppressive cyclosporine A derivative, O-hydroxyethyl-D(Ser)-cyclosporine (SDZ IMM 125), was examined for its ability to induce apoptosis in rat hepatocytes cultured for 4 or 20 h. Four hours after SDZ IMM 125 treatment, chromatin condensation and fragmentation, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeled and Annexin V-positive cells increased dose dependently without any observable lactate dehydrogenase leakage. The activity of the cysteine protease, caspase-3, was increased, but not that of caspase-1 and -6. The specific caspase-3 inhibitor, Ac-Asp-Glu-Val-Asp-aldehyde, inhibited caspase-3 activation and attenuated SDZ IMM 125-induced apoptosis and lactate dehydrogenase leakage. After 20 h of SDZ IMM 125 incubation, the parameters of apoptosis were further increased. Decreased mitochondrial membrane potential (measured by rhodamine 123 uptake) and cytochrome c release went in parallel with ultrastructural mitochondrial changes, and might be regarded as early events that trigger the apoptotic cascade. Transmission electron microscopy showed cytoplasmic blebbing after 4 h of SDZ IMM 125 incubation. As observed by transmission electron microscopy, treatment with SDZ IMM 125 resulted in an increase in the number of necrotic cells after 20 h, but not after 4 h. Our findings suggest that in rat hepatocyte cultures, SDZ IMM 125 is a specific inducer of apoptosis after short-term incubation, and this overlaps with necrosis after longer treatment periods. It is very likely that the necrosis occurring later is the result of the early apoptotic events. The O-hydroxyethyl-D(Ser)-cyclosporine A derivative O-hydroxyethyl-D(Ser)-cyclosporine (SDZ IMM 125), is almost equipotent to cyclosporine A (CsA) as concerns its immunosuppressive properties, i.e., suppression of lymphokine production; however, in animal studies, it possesses a wider therapeutic window. The immunosuppressive effect of both compounds derives from their inhibition of interleukin-2 (IL-2) synthesis in T-helper cells at the mRNA level, thus inhibiting the maturation of cytotoxic T cells. In the T cell, cyclosporines form complexes with immunophilins and inhibit the original peptidyl-prolyl-cis-trans isomerase activity. Because a nonimmunosuppressive CsA analog also binds to and inhibits cyclophilin, the inhibition of this enzyme activity alone does not explain the immunosuppressive properties of CsA and SDZ IMM 125. The immunosuppressant-immunophilin complexes also bind to and inhibit the calciumactivated phosphatase, calcineurin. This could result in an altered modification pattern of cytoplasmic components of transcription factors, thereby disturbing the nuclear translocation, which is a prerequisite for proper IL-2 transcription. As a sequel, the biochemical cascade that transduces activation signals from the T-cell receptor on the surface of the cell to its nucleus is interrupted (Baumann et al., 1992; Borel et al., 1996). In preclinical studies, SDZ IMM 125 caused less renal dysfunction in the rat, compared with CsA (Donatsch et al., 1992; Hiestand et al., 1992). SDZ IMM 125 inhibited the uptake and secretion of bile acids in rat hepatocytes, and, in the isolated perfused rat liver, also bile flow (Wolf et al., 1998). The drug was well tolerated in healthy volunteers, and in psoriatic patients, in whom it had a dose-related beneficial effect in clearing psoriasis (Witkamp et al., 1995). Clinical adverse effects were similar to those reported for CsA, i.e., transient impairment of liver function, which manifests itself by elevated, serum bile-acid levels, together with hyperbilirubinemia. There was clear evidence for liver intolerance of SDZ IMM 125, which resulted in significant dose-dependent increases in the liver-specific serum transaminases. Elevation of the aminotransferases was found more frequently than after treatment with CsA (Witkamp et al., 1995). Received for publication July 29, 1999. 1 Current address: University of Kaiserslautern, Department of Chemistry, Kaiserslautern, Germany. ABBREVIATIONS: CsA, cyclosporine A; TdT, terminal deoxynucleotidyl transferase; Ac, acetate; AMC, 7-amino-4-methyl coumarin; CHO, aldehyde; DEVD, Asp-Glu-Val-Asp; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; LDH, lactate dehydrogenase; SDZ IMM 125, O-hydroxyethylD(Ser)-cyclosporine; TEM, transmission electron microscopy; TUNEL, TdT-mediated dUTP-biotin nick end labeling; VEID, Val-Glu-Ile-Asp; WME, William’s medium E; YVAD, Tyr-Val-Ala-Asp. 0022-3565/00/2931-0024$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 293, No. 1 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 293:24–32, 2000 24 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from In hepatocyte primary cultures and in the isolated perfused liver, SDZ IMM 125 caused the release of lactate dehydrogenase (LDH), which correlated very well with that of serum transaminases (Wolf et al., 1998). In addition, we have shown that CsA and SDZ IMM 125 caused oxidative stress, and that cyclosporine cytotoxicity can be inhibited by antioxidants (Wolf and Donatsch, 1990; Wolf and Broadhurst, 1992; Wolf et al., 1994, 1997; Trendelenburg, 1995). SDZ IMM 125 enhanced the uptake of Ca into liposomal vesicles (Wolf et al., 1998). In the current literature, oxidative stress and increased intracellular Ca concentrations have been described as inducers of apoptosis (Buttke and Sandstrom, 1994), which suggests the potential role of SDZ IMM 125 as an inducer of apoptosis. However, neither inhibition nor induction of apoptosis by SDZ IMM 125 in the liver has been described after in vivo treatment. In contrast, CsA has recently been shown to induce apoptosis in rat hepatocyte cultures treated for 4 and 20 h (Grub et al., 2000). This study was designed to investigate the mechanisms underlying the hepatic side effects of SDZ IMM 125 in relation to apoptosis and necrosis. Morphological and biochemical parameters were determined under in vitro conditions in which specific hepatocellular functions are preserved for up to 20 h. Materials and Methods Animals. Permission for animal studies was obtained from the Veterinäramt Basel-Landschaft, CH-4410 Liestal, and all study protocols were in compliance with institutional guidelines. Male Han Wistar rats were obtained from Biological Research Laboratories (CH-4414 Füllinsdorf, Switzerland). They were kept in Macrolon cages with wood shavings as bedding under optimal hygienic conditions, at a temperature of 22–23°C, a relative humidity of 50 to 74%, and fluorescent light for a 12-h light/dark cycle. They were given water and rodent pellets ad libitum. Hepatocyte Isolation and Cell-Culture Conditions. Rat hepatocytes (rats 180–220 g) were isolated according to the two-step liver perfusion method (Boelsterli et al., 1993). The cells were seeded in 35-mm six-well Primaria culture dishes (Becton Dickinson, Basel, Switzerland) at a density of 0.7 3 10 cells in 2 ml of William’s medium E (WME), or in 60-mm culture dishes (Primaria; Becton Dickinson) at a density of 2 3 10 cells in 5 ml of WME (Life Technologies AG, Basel, Switzerland). The culture medium contained 10% fetal calf serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10 M insulin, and 10 M dexamethasone. After an attachment period of 2 h at 37°C in a 5% CO2, 95% air atmosphere, the medium was changed. The test compound was added together with the new medium. SDZ IMM 125 (Novartis, Basel, Switzerland) was dissolved in dimethyl sulfoxide (DMSO), and this solution was added to the culture medium, which resulted in a final concentration of 1% DMSO in the culture medium. Control plates received the DMSOcontaining medium without SDZ IMM 125. The maximum soluble SDZ IMM 125 concentration was 50 mM. All experiments were checked for the absence of SDZ IMM 125 precipitations. Transmission Electron Microscopy (TEM). The cell cultures were fixed with 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 h or overnight at 4°C. Then, after fixing with 1% OsO4 in 0.1 M cacodylate buffer, pH 7.4, for 1 h at 4°C, the cell cultures were dehydrated in graded ethanol solutions and embedded in Epon according to the method of Pease (1984). Ultrathin sections of hepatocyte cultures from at least two selected tissue blocks per well were counterstained with uranyl acetate (Ac) and lead citrate, and were examined with a Philips CM10 transmission electron microscope. Ultrastructural alterations in the cell cultures treated such as apoptotic bodies, blebs, dilatation of the endoplasmic reticulum, or alterations in the mitochondria (membranes weakly visible, matrix dark, size, and number) were expressed by different scores, depending on the degree of intensity. The following scores were used: 1) alterations observed in one cell per mm 5 marginal; 2) alterations in two cells per mm 5 slight; 3) alterations in three to nine cells per mm 5 moderate; and 4) alterations in ten or more cells per mm 5 marked. Determination of Cytotoxicity. LDH activity in the culture media, as an index of plasma-membrane damage and loss of membrane integrity, was measured spectrophotometrically (Wedler and Acosta, 1994). Enzyme activity was expressed as the percentage of extracellular LDH activity of the total LDH activity on the plates. Determination of Chromatin Condensation and Degradation. Chromatin condensation and fragmentation were determined by Feulgen staining and using light microscopy to count the percentage of cells containing alterations in the nuclear structure (Lillie and Fullmer, 1976). Hepatocyte samples were treated as follows: after the cell culture medium was removed, the cells were fixed overnight with 4% formaldehyde in PBS. After hydrolyzing in 5 N HCl for 90 min at room temperature, the cells were rinsed in distilled water and stained for 30 min in Schiff reagent (Merck, Dietikon, Switzerland). After this, the hepatocytes were rinsed in 0.05 M Na2S2O3 solution for 2 min, washed first in tap water, and then washed in distilled water, and mounted by Crystal Mount (Biomeda, Foster City, CA). After staining, hepatocyte nuclei were violet in color. The following criteria were used: normal nuclei were those in which the chromatin was unaltered and uniformly spread over the whole nucleus. Condensed chromatin was located at the periphery of the nuclear membrane and appeared in a half-moon form. Fragmented chromatin was identifiable by its scattered, drop-like structure, which was located on the area of the original nucleus. When compared with intact cells, the total size of apoptotic nuclei appeared to be smaller and more shrunken. For each sample, 1000 to 1500 nuclei were counted. Determination of DNA Fragmentation. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay was performed using the DNA fragmentation kit TdT-FragEL (Calbiochem, Lucerne, Switzerland). The principle of the assay is based on the fact that TdT binds to exposed 39-OH ends of DNA fragments generated in response to apoptotic signals, and catalyzes the addition of biotin-labeled and unlabeled deoxynucleotides (Gavrieli et al., 1992). Biotinylated nucleotides were detected using a streptavidin-horseradish peroxidase conjugate. Diaminobenzidine reacts with the labeled sample to generate an insoluble, colored substrate at the site of DNA fragmentation. Nonapoptotic cells do not incorporate significant amounts of labeled nucleotide because they lack an excess of 39-OH ends. Nonapoptotic cells were counterstained with methyl green. Positive TUNEL staining was indicated by a dark brown diaminobenzidine signal, whereas shades of bluegreen signified a nonreactive cell. Determination of Membrane Phosphatidylserine Distribution. Phosphatidylserine distribution was detected by labeling the cells with the biotin conjugate of Annexin V (Roche, Basel, Switzerland) according to the method of Vermes et al. (1995). Cells were washed with binding buffer (HEPES/NaOH 10 mM, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and incubated for 1 h with Annexin-biotin 1:20 in binding buffer. After washing in PBS, the cells were fixed with Formalin. To detect Annexin V, cells were washed in PBS and incubated with Streptavidin peroxidase complex (Elite Kit Vector; Vector, Burlingame, CA) for 30 min. After another washing, the ABC substrate (Biomeda) was added for 5 to 15 min. Cells were washed and mounted with Crystal Mount (Biomeda). Annexin-positive cells were detected by their brown color. Determination of Caspase-1, -3, and -6 Activity. Caspase activity was determined according to the method of Rodriguez et al. (1996). After incubation, 2 3 10 cells were washed once in ice-cold 2000 SDZ IMM 125 Induces Apoptosis in Rat Hepatocytes 25 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from PBS and lysed in 1 ml of buffer A [10 mM HEPES, pH 7.4, 42 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), and protease inhibitors (Complete; Roche, Basel, Switzerland)]. After three thaw-freeze cycles, the lysate was centrifuged for 20 min at 13,000g at 4°C. The supernatant (lysate) was removed and stored at 280°C until the assay was performed. Lysates (70 mg protein) were assayed in 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS; Sigma, Buchs, Switzerland), 100 mM HEPES, 10% sucrose, and 10 mM DTT, pH 7.5, with or without protease inhibitors (100 mM); caspase-1 [Ac-TyrVal-Ala-Asp (YVAD)-aldehyde (CHO)], caspase-3 [Ac-Asp-Glu-ValAsp (DEVD)-CHO], or caspase-6 [Ac-Val-Glu-Ile-Asp (VEID)-CHO] (Bachem, Bubendorf, Switzerland) were added in DMSO. The reaction was started with 20 mM the substrate for caspase-1 [Ac-YVAD7-amino-4-methyl coumarin (AMC)], caspase-3 (Ac-DEVD-AMC), and caspase-6 (Ac-VEID-AMC), which were labeled with the fluorochrome, AMC (Bachem, Bubendorf, Switzerland), and the reaction was followed for 60 min. Fluorescence was measured at excitation 360 nm and emission 460 nm in a fluorescence plate reader. Fluorescence intensity was calibrated with standard concentrations of AMC. Protease activity was calculated from the slope of the recorder trace and expressed as picomoles per milligrams protein per min. The difference between the substrate cleavage activity levels in the presence and absence of selective inhibitors reflected the contribution of the activity of either caspase-1, -3, or -6 enzyme. Determination of Mitochondrial Membrane Potential. Mitochondrial membrane potential was determined by the uptake of rhodamine 123 according to the method of Wu et al. (1990). After treatment, hepatocytes cultured on 96-well plates (Primaria; Becton Dickinson), were washed in PBS and incubated with 10 mg/ml rhodamine 123 (Molecular Probes, Leiden, the Netherlands) for 30 min at 37°C. After additional washing, the hepatocytes were incubated with WME for 30 min. Ethanol/water 1:1 was used to extract the Fig. 1. Effect of SDZ IMM 125 on release of LDH after 4 (M) and 20 h (f). Data are expressed as mean 6 S.D. (n 5 3). Statistically significant differences versus the control group are expressed as ***P , .001. Fig. 2. Effect of SDZ IMM 125 on chromatin condensation in hepatocytes after 4 (M) and 20 h (f). Data are expressed as mean 6 S.D. (n 5 3). Statistically significant differences versus the control group are expressed as **P , .01 and ***P , .001. Fig. 3. Effect of SDZ IMM 125 on chromatin fragmentation in hepatocytes after 4 (M) and 20 h (f). Data are expressed as mean 6 S.D. (n 5 3). Statistically significant differences versus the control group are expressed as **P , .01 and ***P , .001. Fig. 4. Effect of SDZ IMM 125 on TUNEL-positive hepatocytes after 4 (M) and 20 h (f). Data are expressed as mean S.D. (n 5 3). Statistically significant differences versus the control group are expressed as *P , .05, **P , .01, and ***P , .001. 26 Grub et al. Vol. 293 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from