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TOXICOLOGY

Apoptosis in Microencapsulated Juvenile Rabbit Chondrocytes Induced by Ofloxacin: Role Played by ₁-Integrin Receptor

National Beijing Center for Drug Safety Evaluation and Research, Beijing Institute of Pharmacology and Toxicology, Beijing, China (Z.S., S.P., C.W., H.L., Y.W., Q.L., M.L., Y.D., G.H.); and Neuroscience Program, Michigan State University, East Lansing, Michigan (R.K.H.)

Received December 7, 2006; accepted March 29, 2007.

	Abstract

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Quinolone(s) (QNs) is widely used in infection therapy due to its good antimicrobial characteristics. However, QNs-induced arthropathy of immature animals has led to restrictions on the therapeutic use of these antimicrobial agents. The exact mechanism(s) of QNs-induced chondrotoxicity remain unknown. In the present study, we investigated the possible mechanism of ofloxacin (one typical QNs)-induced injuries of chondrocytes. Juvenile rabbit joint chondrocytes cultured in alginate microspheres were incubated with ofloxacin at concentrations of 0, 2, 5, 10, 20, and 40 µg/ml for up to 96 h. Concentration of 10 µg/ml ofloxacin induced apoptosis of chondrocyte with visible apoptotic signs, including degradation of poly(ADP-ribose) polymerase, caspase-3 activation, and DNA ladder formation. Furthermore, extracellular signal-regulated kinase 1/2 (phospho-ERK1/2) and growth factor receptor-bound protein 2 (Grb2) were significantly reduced, and similar changes were also observed in the ₁-integrin receptor as assessed by immunoblotting. However, the mRNA level of ₁-integrin obtained from reverse transcription-polymerase chain reaction remained unchanged. Results of ₁-integrin immunoprecipitation have also shown that ₁-integrin did not interact with activated intracellular signaling proteins. In addition, ofloxacin did not induce apoptosis and decrease ₁-integrin expression in chondrocytes supplemented with Mg²⁺, and the ofloxacin-induced apoptosis was caspase-8-dependent, inhibition of which did not affect the expression mode of phospho-ERK1/2 and ₁-integrin. Our results demonstrate that ofloxacin affects ₁-integrin receptor functions and the ERK mitogen-activated protein kinase signaling pathway, causing caspase-8-dependent apoptosis after exposure of 48 h.

However, the exact mechanism of QNs-induced arthropathy is still unknown. Recently, a number of studies have demonstrated that QNs-induced arthropathy is possibly due to the magnesium-chelating properties of QNs (Stahlmann et al., 1995; Shakibaei et al., 1996; Vormann et al., 1997). QNs are metal chelators (Lomaestro and Bailie, 1991), and one possible consequence of the chelation of Mg²⁺ can be an effect on integrin, the binding of which to their ligands is a cation-dependent process (Dickeson et al., 1997). Integrins are important surface molecules on chondrocytes and transmit signals in both directions across the plasma membrane (Akiyama, 1996), thus regulating cell adhesion, proliferation, differentiation, and proteoglycan synthesis.

The interaction between chondrocytes and matrix proteins is mediated largely by the ₁ subfamily of integrins (Dürr et al., 1993; Woods et al., 1994). Therefore, QNs appear to disturb the normal functioning of signaling proteins like ₁-integrin and, subsequently, their essential regulation of cell-cell and cell-matrix interactions.

The activation of specific intracellular signaling proteins, due to interactions between matrix proteins and integrins, has been studied in various cell types. Shakibaei et al. (2001) demonstrated that MAPK signaling pathway has regulatory functions on differentiation and survival of chondrocytes and that inhibition of this pathway can induce apoptosis of human chondrocytes. Recently, Sendzik et al. (2005) have shown that changes in ₁-integrin receptor protein and its downstream signaling proteins (such as phospho-ERK1/2) result in apoptosis of tenocytes, which is considered as the final event in QNs-induced tendopathies. Parallels might exist in the mechanisms of chondrotoxicity and tendotoxicity of QNs, because tendon and cartilage have marked organizational similarities; both are characterized by a low vascularization (bradytrophia tissue) and similar matrix components, transmembrane and intracellular signaling proteins.

The observations mentioned above led us to believe that disturbance of ₁-integrin receptor functions, subsequently inactivating the ERK/MAPK signaling pathway, might induce chondrocyte apoptosis. In addition, there is no report so far showing the involvement of apoptosis in QNs-induced joint cartilage injury. Therefore, the aim of the present study was to investigate whether QNs induce chondrocyte apoptosis and the possible role of ₁-integrin receptors in the process.

Because signaling events associated with ₁-integrin seem to take place only inside the three-dimensional (3-D) collagen matrix, rather than in monolayer cultures (Heino, 2000), a 3-D cell culture system with chondrocytes microencapsulated in alginate microspheres was employed as an experiment model.

	Materials and Methods

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Materials. All chemicals (ofloxacin: CAS 82419-36-1; C₁₈H₂₀FN₃O₄) were of reagent grade or better and were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Sodium alginate (type -hydroxybutyrate) was obtained from Kelco (Waterfield, UK). Acridine orange (AO, 10 mg/ml) and ethidium bromide (EB, 10 mg/ml) were purchased from Invitrogen (Carlsbad, CA), and further dilutions were made in 0.9% normal saline before use. The caspase-3 inhibitor z-DEVD-fmk was purchased from MBL International (Woburn, MA) as sterile solution (2 mM). RNA isolation kits, Gene-Amp RNA PCR kit, and GAPDH primers were from Invitrogen. The primary rabbit monoclonal antibody against ₁-integrin was purchased from BOSTER (Wuhan, China), and mouse monoclonal antibodies against uncleaved (116 kDa) and cleaved (85 kDa) PARP, caspase-3, type I collagen, type II collagen, Grb2, total-ERK1/2, and phospho-ERK1/2 were from Cell Signaling (Beverly, MA). Polyclonal antibody against the ₁-cytoplasmic domain was provided by our laboratory. Goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase was from BOSTER. Suicide Track DNA Ladder Isolation Kit (AM41-1EA) was from EMD Biosciences (San Diego, CA).

Cell Culture and Microencapsulation. All tissue manipulations were carried out under sterile, aseptic conditions. Juvenile New Zealand White rabbit (21-day-old) joint cartilage segments were excised from both humeral and femoral heads and placed in Dulbecco's modified Eagle's medium (DMEM) solution containing 200 µg/ml gentamycin and 0.5 µg/ml amphotericin immediately after surgery. The perichondrium was removed to prevent contamination with fibroblasts, and minced cartilage particles (1 mm³) were digested with 0.15% collagenase II solution in fresh DMEM at 37°C for 8 to 12 h. The resulting suspension was centrifuged at 400g for 10 min, and the chondrocyte pellets were resuspended in DMEM with 146 µg/ml L-glutamine, 50 µg/ml gentamycin, and 10% fetal bovine serum. Chondrocytes were plated on 75-cm² vented polystyrene cell culture flasks and incubated in a humidified incubator at 37°C with 5% CO₂. The culture medium was changed twice a week. Cells were trypsinized using trypsin/EDTA solution (0.05/0.02%) for 2 to 5 min at 37°C and serially passaged after reaching confluence.

One volume of chondrocytes suspension (2.5 x 10⁶ ml^–1 viable cells) was mixed gently with one volume of sodium-alginate solution [alginate 0.5–3% (w/v) in PBS] to give a cell density of 1.25 x 10⁶ ml^–1 viable cells in 0.25 to 1.5% -hydroxybutyrate alginate as shown under Results. An air jet pellator was used to produce microdrops of a defined diameter (400–500 µm) using a flow rate of 62 ml h^–1 and a pressure of 190 ± 10 mbar. The alginate microdrops were collected in a polymerization buffer [620 ml of glucose solution, 100 ml of HEPES-buffer (250 mM HEPES adjusted to pH 7.6 with NaOH), 100 ml of salt solution (1 M NaCl, 23 mM KCl, and 1 M CaCl₂·2H₂O), 150 ml of amino acid solution, 10 ml of glutamine solution, 5 ml of insulin solution, and 2 µg/ml bovine serum albumin] for microencapsulation. During microencapsulation, the polymerization buffer was constantly stirred. The resulting beads were removed from the polymerization buffer and washed with PBS. Thereafter, the beads were transferred to the cell culture medium.

Microencapsulated chondrocyte beads were placed in 14.5-cm tissue culture dishes at a density of 1.5 x 10⁷ cells per dish in 30 ml of culture medium consisting of William's medium E supplemented with 10% fetal bovine serum, 7 µM insulin (I-6634; 28.5 IU mg^–1 insulin), 100 nM dexamethasone, 4 mM L-glutamine, and 1% penicillin/streptomycin solution. After 3 h, the culture medium was replaced by 30 ml of fresh medium.

Ofloxacin Treatment. After 24 h of culture, the medium was renewed. The microencapsulated chondrocytes were exposed to ofloxacin at 0 to 40 µg/ml for up to 96 h. To make ofloxacin solutions achieve optimal solubility and final pH at 7.2, ofloxacin was prediluted in a NaOH solution (0.1 N), followed by dilution in PBS solution at pH 5 to 6. The cells were cultured in a humidified incubator (5% CO₂) at 37.5°C. Care was taken to distribute the beads evenly on the plate through the duration of the experiment.

Harvesting of Ofloxacin-Treated Microencapsulated Chondrocytes. After incubation with ofloxacin, the beads were transferred to 50-ml tubes on ice. Two volumes of sodium citrate buffer (10 mM trisodium citrate in PBS, pH 7.4, at 4°C) were added to one volume of beads. The beads were gently shaken and dissolved within 2 min. Three volumes of PBS (4°C) were added to the cell suspension, and the cells were harvested by centrifugation (50g, 10 min, 4°C). The pellets were resuspended in 1 ml of PBS and homogenized by sonication.

Cell Viability Assay and Determination of Apoptosis. Cell viability was determined by a modified 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (Mosmann, 1983). At the end of treatment, 100 µl of MTT (5 mg/ml in 1 M PBS, pH 7.6) was added to 1 ml of microencapsulation culture solution (approximately five to seven microcapsules) to each well of six-well plates and allowed to incubate for 2 h at 37°C in 5% CO₂/95% air. The MTT solution was then removed, and 100 µlof N,N-dimethylformamide destaining solution (N,N-dimethylformamide: Triton X-100, 3:1; w/w) was added. The plates were agitated on a plate shaker for 10 min at room temperature to thoroughly dissolve the MTT product, and 0.2 ml was transferred to 96-well plates. The optical density (OD) was measured using a microplate reader at 570 nm. Viability of the cells without ofloxacin treatment was defined as 100%, and the experiment was repeated three times.

Quantitation of apoptosis was performed using fluorescent staining as described previously (Duke and Cohen, 1992). Dual staining allows separate enumeration of viable nonapoptotic, viable (early) apoptotic, nonviable (late) apoptotic, and necrotic cells. Viable cells have uniformly stained green nucleus. Viable (early) apoptotic cells have condensed or fragmented green nuclei. All nonviable cells have orange nuclei, because ethidium bromide staining overwhelms acridine orange staining. Nonviable (late) apoptotic cells have orange-fragmented nuclei, whereas necrotic cells have uniformly stained nonfragmented orange nuclei. Cells were pelleted, resuspended in 50 µl of DMEM, and stained with 4 µl of AO-EB working solution (100 µg/ml of acridine orange and 100 µg/ml of ethidium bromide in 0.9% saline). Samples were immediately analyzed by fluorescence microscopy utilizing the fluorescein filter (excitation at 488 nm, emission at 520 nm). A minimum of 180 cells was counted in multiple, randomly selected fields from each sample. Results were expressed as mean percentage of apoptosis in different fields ± S.D.

Measurement of Proteoglycan Content. Proteoglycan (PG) content was assessed by 1,9-dimethylmethylene blue assay (DMB; Polysciences, Warrington, PA). Chondrocyte/alginate beads/cartilage explants were harvested and dissolved, and the two compartments [cell-associated matrix and further removed matrix] were separated by centrifugation at 100g for 10 min at 4°C. Each fraction was digested with papain (concentration of papain: cell-associated matrix, 20 mg/ml; further removed matrix, 40 mg/ml) at 55°C for 18 h. The digested samples (75 µl) were mixed with 25 µl of 2.88 M GuHCl solution and 200 µl of DMB reagent in a 96-well plate, and absorbance was immediately measured at 590 nm by a plate reader (SPECTRA MAX250; Molecular Devices, Sunnyvale, CA). PG content was determined using a chondroitin-6-sulfate standard curve (0–100 µg/ml). DNA content was determined by the Hoechst 33258 DNA binding assay (Kim et al., 1988). Total amount of PG per well was normalized against the total amount of DNA per well.

Analysis of DNA Fragmentation by Agarose Gel Electrophoresis. Genomic DNA was prepared from 5 x 10⁶ cells and electrophoresed through a 1.5% agarose gel. Oligonucleosomal apoptotic DNA fragments ladder separated on the gels were visualized after EB staining, and photodocumentation was performed using a Fluorichem 8000 fluorimager (Alpha Innotech, San Leandro, CA).

RNA Extraction and RT-PCR. In brief, total RNA in chondrocytes was obtained using a RNA isolation kit (Invitrogen). Cells were washed with sterile PBS and then mixed into 6 ml of a denaturing solution. The solution was transferred to RNase-free centrifuge tubes before sodium acetate was added to a final concentration of 0.2 M. A phenol-chloroform extraction was performed followed by isopropanol precipitation using a routine method (Sambrook et al., 1989). A second RNA extraction with 600 µl of denaturing solution was performed on each RNA pellet followed by a second isopropanol precipitation. The RNA pellets were washed with 75% ethanol and dissolved in 20 µl of RNase-free water. The RNA concentration and purity were calculated from the reading at OD₂₆₀ and OD₂₈₀,as described previously (Sambrook et al., 1989). Aliquots of 10 and 20 µg of RNA with an OD₂₆₀:OD₂₈₀ ratio >1.8 were stored in ethanol at –70°C until use.

The mRNA in 0.5 µg of total RNA was transcribed to cDNA using a Gene-Amp RNA PCR kit (Invitrogen). The final concentrations of reagents for reverse transcription in 1x PCR buffer II were 5 mM MgCl₂, 1 mM of each nucleotide, 1 U/µl RNase inhibitor, 2.5 U/µl reverse transcriptase, and 2.5 µM random hexamers. The reverse transcription reaction was carried out for 10 min at room temperature followed by 45 min at 42°C. After reverse transcription, 10 µlof each sample was removed for PCR amplification in PCR buffer II containing 2 mM MgCl₂, 2.5 U/100 µl Taq polymerase, and 0.3 µMof each ₁-oligonucleotide primer. The ₁ primers were selected from regions highly conserved between human and rabbit ₁-integrin. The ₁-integrin primers used were 5'-GACCTGCCTTGGTGTCTGTGC-3' and 5'-AGCAACCACACCAGCTACAAT-3'; after incubation for 4 min at 95°C, the samples were amplified for 32 cycles at 95°C for 1 min, 54°C for 1 min, and 72°C for 3 min. A final 7-min extension at 60°C was performed after the 32 cycles. GAPDH primers were 5'-CGTCTTCACCACCATGGAGA-3' and 5'-CGGCCATCACGCCACAGTTT-3'. Amplification of control primers for GAPDH was carried out as recommended by the supplier, with a final concentration of 0.4 µM of each primer and temperature cycles of 95°C for 1 min and 60°C for 1 min for 32 cycles followed by a final 7-min extension at 60°C. Amplified sequences were analyzed on 1.6% agarose-ethidium bromide gels. Ratios of all experimental bands to GAPDH were used to measure changes in mRNA expression among the different treatment groups.

Immunoprecipitation and Western Blot Analysis. A detailed description of the technique used for the following experiments has been published previously (Shakibaei et al., 1999). In brief, chondrocytes were harvested from alginate cultures and then plated on dishes coated with collagen type II, poly-L-lysine, and anti-integrin ₁ and then treated with 10 µg/ml ofloxacin for 48 h or left untreated. After rinsing with PBS, treated and untreated cells were extracted with lysis buffer containing 50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 0.01% (v/v) aprotinin, 4 µg/ml pepstatin A, 10 µg/ml leupeptin, and 1 mM phenylmethyl sulfonylfluoride on ice for 30 min. For immunoprecipitation, samples were incubated with primary antibody for the target protein (or IgG control antibody) for 1 to 3 h at 4 °C and then precipitated by incubating with protein A/G-agarose overnight. Pellets were washed once in lysis buffer followed by three washes in wash buffer consisting of 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.2% Igepal CA-630 and then three washes in wash buffer containing 10 mM Tris-HCl, pH 7.5, and 0.2% Igepal CA-630. Immunoprecipitated proteins were analyzed by Western blot. For Western blot analysis, after adjustment to a similar total protein concentration, samples were separated by SDS-polyacrylamide gel electrophoresis (5, 7.5, 10, or 12% gels) under reducing conditions and then transferred onto nitrocellulose membranes. Membranes were preincubated in blocking buffer [5% (w/v) skim milk powder in PBS and 0.1% Tween 20] for 30 min before incubation with primary antibodies for 2 h at room temperature. The membranes then were washed three times in Tris-buffered saline plus Tween 20 to remove unbounded antibodies and then incubated with horseradish peroxidase-conjugated secondary antibody in 3% Tris-buffered saline plus Tween 20for 30 min at 37°C. The immunoreactive bands were visualized using the enhanced chemiluminescence reagents (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to manufacturer's protocol and were quantitated by densitometric scanning of X-ray films and analyzed using Quantity One 4.1.1 software (Bio-Rad, Hercules, CA). Differences and changes in protein expression were determined relative to the internal control GAPDH. GAPDH levels were determined for each condition to verify that equal amounts of protein were loaded in different lanes. In addition, the density of each protein band was normalized to GAPDH to correct for small differences in protein loading among the lanes.

Statistical Analysis. The results of immunoblotting were expressed as the arithmetic means ± S.D. of a representative experiment performed in triplicate. The means were compared using ANOVA followed by a post hoc Dunnett's t test assuming equal variances; p < 0.05 was considered statistically significant. This statistical analysis was performed with treated and control samples after 96 h of incubation only.

	Results

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Morphological Differences in Chondrocytes Cultured in Monolayer and 3-D Microcapsules. Chondrocytes in hyaline cartilage can be identified by a spherical shape in an abundant extracellular matrix. They were responsible for the synthesis, maintenance, and maturation of the matrix within which they were embedded. Figure 1 depicts the representative morphology of chondrocytes cultured in monolayer and 3-D microcapsules after 96 h of culture. Differences in cell morphology were evident between the two types of chondrocyte culture (Fig. 1, A and B). The monolayer cultures produced fibroblast-like chondrocytes exhibiting a flat, elongated morphology, expressed elevated levels of type I collagen, and reduced levels of type II collagen and proteoglycan (Fig. 1, A, C, and D). Microencapsulated chondrocytes maintained round shape and expressed high levels of extracellular matrix molecules: type II collagen and proteoglycan and low levels of type I collagen (Fig. 1, B, C, and D). Expression of type II collagen and proteoglycan in microencapsulated chondrocytes was more similar to that in cartilage explant compared with monolayer chondrocytes (Fig. 1, C and D).

View larger version (58K): [in this window] [in a new window]   Fig. 1. Morphological and functional difference between chondrocytes cultured for 96 h in monolayer and 3-D microcapsules. Rabbit joint chondrocytes in monolayer (A) and cultured in alginate microcapsules (B) in brightfield microscopy. Expression of type I collagen and type II collagen is determined by Western blot analysis (C), and content of proteoglycan is determined by DMB assay (D), as described under Materials and Methods. The results of three experiments are shown as mean ± S.D.

The Effect of Ofloxacin on Chondrocyte Viability Cultured in Monolayer and 3-D Microcapsules. Chondrocytes cultured in monolayer and microcapsules were exposed to ofloxacin in a range of concentrations (0–40 µg/ml) for different times (12–96 h), and the effect of ofloxacin on cell viability was investigated by MTT assay. In both types of chondrocyte cultures, ofloxacin caused a decrease in cell viability in a concentration- and time-dependent manner (Fig. 2, A and B). However, joint chondrocytes cultured in microcapsules were more sensitive to ofloxacin than those in monolayer (Fig. 2B). Loss of viability of chondrocyte cultured in microcapsules began to be more evident compared with monolayer after treatment with 2, 5, 10, 20, and 40 µg/ml ofloxacin at 24 h, which was decreased by 12.9, 14.2, 24.2, 31.9, and 33.6%, respectively (Fig. 2B). At 10 µg/ml ofloxacin, near the human plasma concentration, exposure for 12 to 96 h decreased the microencapsulated chondrocytes viability significantly, compared with chondrocytes cultured in monolayer (Fig. 2C).

View larger version (21K): [in this window] [in a new window]   Fig. 2. Differential effects (C) of ofloxacin on cell viability of rabbit joint chondrocytes cultured in monolayer (A) and alginate microcapsules (B). Cells were exposed to 0, 2, 5, 10, 20, and 40 µg/ml ofloxacin and incubated for up to 96 h. Live (viable) cells were determined by MTT assay, as described under Materials and Methods. The results of three experiments are shown as mean ± S.D. *, p < 0.05 versus 2-D culture.

Ofloxacin Induces Apoptosis in Microencapsulated Chondrocytes. To investigate whether the ofloxacin-induced loss in cell viability was due to apoptosis, microencapsulated chondrocytes were exposed to the increasing concentrations of ofloxacin for 72 and 96 h, and the fraction of dead, apoptotic, and necrotic cells were determined (data of the concentration 20 and 40 µg/ml not shown considering the in vivo plasma concentration of ofloxacin) (Fig. 3). As expected, ofloxacin induced apoptosis of microencapsulated chondrocyte in a concentration-dependent manner. The percentage of dead cells was significantly increased by 20.8, 40.3, and 55.6% for 72 h and 40.9, 65.8, and 84.4% for 96 h, respectively, as the concentration of ofloxacin was increased from 0 to 2, 5, and 10 µg/ml and was higher at 96 h compared with 72 h of exposure to ofloxacin (Fig. 3A). Analysis of the same cell population revealed that the percentage of apoptotic cells increased by 26.4, 41.5, and 57.7% for 72 h and 40.7, 66.7, and 85.9% for 96 h, respectively, in correlation to the percentage of dead cells (Fig. 3B). The fraction of apoptotic cells after 72 and 96 h of exposure to ofloxacin was similar to that of dead cell under the same conditions (Fig. 3, A compared with B). No significant increase in the percentage of necrotic cells was observed in the exposed cells compared with control unexposed cells under these experimental conditions (Fig. 3C). The above results shown in Fig. 3 demonstrate that ofloxacin-induced cell death in microencapsulated chondrocytes was mainly due to apoptosis, rather than necrosis at the concentration of 2, 5, and 10 µg/ml after 72 and 96 h of exposure.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Ofloxacin induces apoptosis in rabbit joint chondrocytes in a concentration- and time-dependent manner. Cells were exposed to 0, 2, 5, and 10 µg/ml ofloxacin and incubated for 72 and 96 h. The percentage of dead (A), apoptotic (B), and necrotic (C) cells was determined by fluorescent staining, as described under Materials and Methods. The results of three experiments are shown. Bars represent the mean ± S.D. (*, p < 0.05).

To further confirm the occurrence of ofloxacin-induced apoptosis, microencapsulated chondrocytes were incubated with ofloxacin at 0, 5, and 10 µg/ml for 96 h. PARP cleavage and DNA fragmentation, characteristic of apoptotic cells, then were analyzed. PARP is a zinc-finger DNA-binding protein, which catalyzes the synthesis of poly(ADP-ribose) from its substrate -NAD⁺ and can be selectively cleaved by caspase-3 during apoptosis (Tewari et al., 1995; Salvesen and Dixit, 1997). Therefore, the PARP cleavage has been regarded as evidence of caspase activation and widely used as a hallmark of cell apoptosis.

In the present study, the native uncleaved 116-kDa PARP band in control, unexposed cells were increasingly cleaved to the 85-kDa form when the cells were exposed to increasing concentrations of ofloxacin from 5 to 10 µg/ml (Fig. 4A). Under these conditions, GAPDH levels remained unchanged in the same samples, confirming the absence of experimental artifacts. In addition, one important characteristic feature of apoptosis was the fragmentation of intact DNA into internucleosomal fragmentation to fragments of 180 base pairs and multiples, resulting in a ladder in gel electrophoresis. As shown in Fig. 4B, ofloxacin induced internucleosomal DNA fragmentation, clearly exhibiting the characteristic ladder of oligonucleosomal DNA fragments, which was not observed in unexposed control cells. Collectively, the detection of apoptosis by PARP cleavage and the characteristic DNA ladder confirm the occurrence of apoptosis in microencapsulated chondrocytes exposed to ofloxacin.

View larger version (54K): [in this window] [in a new window]   Fig. 4. PARP cleavage and DNA fragmentation are observed in rabbit joint chondrocytes undergoing ofloxacin-induced apoptosis. Cells exposed to 0, 5, and 10 µg/ml ofloxacin for 96 h were analyzed for PARP cleavage by Western blot (A), as described under Materials and Methods. The native 116-kDa form and the cleaved 85-kDa form of PARP were detected by anti-PARP antibody. GAPDH levels were determined to normalize for the quantity of protein retained on the blot. Cells were exposed to 0, 2, 5, and 10 µg/ml ofloxacin for 96 h, and DNA fragmentation was analyzed as described under Materials and Methods (B). Size markers (M) are shown for demonstrating the characteristic DNA ladder.

Ofloxacin Stimulates Activation of Caspase-3. Caspases are common apoptotic cell death executors. To determine whether caspases were activated in the presence of ofloxacin, we assessed the activation of caspase-3, known as the final effector of apoptosis (Saraste and Pulkki, 2000), by analyzing the processing and activation of caspase-3. Microencapsulated chondrocytes were exposed to 0 or 10 µg/ml ofloxacin for 96 h, and the activation of caspase-3 was determined by Western blot analysis. Figure 5A shows that the level of the active enzyme complex, the p19 smaller subunit, was significantly increased in exposed cells compared with control unexposed cells. To further confirm whether activation of caspase-3 was involved in ofloxacin-induced apoptosis, the effect of the caspase-3 inhibitor z-DEVD-fmk on ofloxacin-induced apoptosis was investigated. Microencapsulated chondrocytes were exposed to 0 or 10 µg/ml ofloxacin for 96 h in the presence or absence of 2 µM z-DEVD-fmk, and cells were scored for apoptosis by fluorescent staining (Fig. 5B). z-DEVD-fmk reduced ofloxacin-induced apoptosis by approximately 80%, which exhibited the same effect on apoptosis of the control-unexposed cells. Higher concentration (5 µM) of z-DEVD-fmk was not any more effective in inhibiting ofloxacin-induced apoptosis (data not shown), indicating that maximal inhibition of caspase-3 was achieved under these experimental conditions. These results indicated that caspase-3, in particular, was involved in ofloxacin-induced apoptosis.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Effect of ofloxacin on caspases-3 activation. Cells exposed to 0 or 10 µg/ml ofloxacin for 96 h were prepared and analyzed for caspases-3 processing by Western blot with an anti-caspase-3 antibody, as described under Materials and Methods. The unprocessed form of caspase-3 (procaspase-3; 32 kDa) and the cleavage product (p19) of the active enzyme are indicated (A). Cells were preincubated with 0 or 2 µM caspase-3 inhibitor z-DEVD-fmk for 1 h, followed by exposure to 0 or 10 µg/ml ofloxacin for 96 h. Inhibition of ofloxacin-induced apoptosis by caspase-3 inhibitor was analyzed by fluorescent staining (B), as described under Materials and Methods. Results from three experiments are shown; bars represent the mean ± S.D. (*, p < 0.05).

Effect of Ofloxacin on ₁-Integrin Function and ERK/MAPK Signaling Pathway. To further explore whether ofloxacin-induced apoptosis was related to ₁-integrin receptor and ERK/MAPK signaling pathway, which are known to be involved in apoptosis of chondrocytes (Shakibaei et al., 1999, 2001), Western blot analysis was used to determine the effects of ofloxacin on the expression of ₁-integrin, Grb2, and phospho-ERK1/2. Microencapsulated chondrocytes were incubated with 10 µg/ml ofloxacin for 12, 24, 48, 72, and 96 h. RT-PCR analysis using primers for ₁-integrin revealed no significant difference (Fig. 6A). However, ofloxacin caused a decrease in the receptor chain ₁-integrin (117 kDa) under these experimental condition after 48 h of exposure (Fig. 6B). Therefore, we further assessed the effect of ofloxacin on ₁-integrin function by coimmunoprecipitation assays. As shown in Fig. 7, the cytoplasmic domain of ₁-integrin did not interact with activated intracellular signaling proteins (FAK, vinculin, paxillin, and -actinin) after exposure of cells to 10 µg/ml for 48 h. Densitometric analysis of a representative experiment, performed in triplicate from ofloxacin-treated chondrocytes, showed that the relative ratios of paxillin, -actinin, vinculin, and FAK expression to the ₁-integrin pulled down by immunoprecipitation with anti-₁-integrin antibody decreased by 94, 97, 81, and 92% compared with untreated chondrocytes, respectively. In addition, it is found that ofloxacin did not affect the occurrence of apoptosis and ₁-integrin expression of microencapsulated chondrocytes under the condition of magnesium supplement (Fig. 8). The signaling proteins Grb2 (23kDa) (Fig. 9A) and phospho-ERK1/2 (44/42 kDa) (Fig. 9B) were reduced after exposure to 10 µg/ml ofloxacin for postexposure 48, 72, 96 h, and ofloxacin also markedly decreased the level of phospho-ERK1/2 and ₁-integrin for the same time points in the presence of specific caspase-3 inhibitor z-DEVD-fmk (Fig. 10). In addition, an inhibitor of the ERK/MAPK signaling pathway, U0126, caused apoptosis of microencapsulated chondrocytes in a concentration-dependent manner (Fig. 11A). However, levels of ₁-integrin protein were unchanged under the same conditions (Fig. 11B). These data suggested that ofloxacin probably inactivated the ERK/MAPK signaling pathway of microencapsulated chondrocytes by disturbing the function of ₁-integrin receptors.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Effects of ofloxacin on gene and protein expression of 1-integrin. RT-PCR and Western blot results obtained with cells exposed to 10 µg/ml ofloxacin and incubated for 12, 24, 48, 72, and 96 h, as described under Materials and Methods. The 1-integrin mRNA levels were analyzed by RT-PCR and normalized against GAPDH (A). An antibody against 1-chain of integrins was used for detection of the protein expression levels by Western blot (B). The results of three experiments are shown; bars represent the mean ± S.D. *, p < 0.05 versus controls after 96 h of incubation (ANOVA followed by a post hoc Dunnett's t test).

View larger version (31K): [in this window] [in a new window]   Fig. 7. Effects of ofloxacin on 1-integrin function. A, serum-starved cells were either treated with 10 µg/ml ofloxacin or left untreated for 48 h and then immunoprecipitated (IP) with anti-1-cytoplasmic domain (1) antibody and normal IgG serum control (C). Immunoprecipitates of the indicated proteins were separated by SDS-polyacrylamide gel electrophoresis and probed with antibodies specific for paxillin, -actinin, vinculin, and FAK, as described under Materials and Methods. IgG control antibodies were used to control for nonspecific interaction. Results shown are a representative of three independent experiments. IgH, immunoglobulin heavy chain. B, immunoprecipitation blots from ofloxacin-treated and untreated chondrocytes were probed with anti-1-cytoplasmic domain antibody and quantified by densitometry.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Effects of ofloxacin on 1-integrin expression and apoptosis in medium supplemented with magnesium. Western blot and fluorescent staining results were obtained with cells exposed to 10 µg/ml ofloxacin and incubated for 12, 24, 48, 72, and 96 h in medium supplemented with 97.7 µg/ml magnesium, as described under Materials and Methods. An antibody against 1-chain of integrins was used for detection of the protein expression levels by Western blot (A). The quantitation of apoptosis was analyzed by AO-EB fluorescent staining assay (B). The results of three experiments are shown; bars represent the mean ± S.D. *, p < 0.05 versus controls after 96 h of incubation (ANOVA followed by a post hoc Dunnett's t test).

View larger version (27K): [in this window] [in a new window]   Fig. 9. Effects of ofloxacin on Grb2 adaptor (A) and phosphorylated ERK1/2 protein (B) expression. Cells were treated with 10 µg/ml ofloxacin for up to 96 h as indicated. An antibody against Grb2 adaptor, phospho-ERK1/2, and total-ERK1/2 was used for detection of the protein by Western blot, respectively, as described under Materials and Methods. The results of three experiments are shown; bars represent the mean ± S.D. *, p < 0.05 versus controls after 96 h of incubation (ANOVA followed by a post hoc Dunnett's t test).

View larger version (24K): [in this window] [in a new window]   Fig. 10. Effects of ofloxacin on phosphorylated ERK1/2 (A) and 1-integrin (B) expression in the presence of specific caspase-3 inhibitor z-DEVD-fmk. Cells were treated with 10 µg/ml ofloxacin for up to 96 h after pretreatment with 4 µM z-DEVD-fmk for 1 h. The antibodies against phospho-ERK1/2, total-ERK1/2, and 1-integrin were used for detection of the protein by Western blot, as described under Materials and Methods. The results of three experiments are shown; bars represent the mean ± S.D. *, p < 0.05 versus controls after 96 h of incubation (ANOVA followed by a post hoc Dunnett's t test).

View larger version (18K): [in this window] [in a new window]   Fig. 11. Effects of MAPK kinase 1/2 inhibitor U0126 on apoptosis and 1-integrin expression. Cells were incubated with the increasing concentrations of U0126. Apoptosis was assessed by fluorescent staining assay (A), and an antibody against 1-chain of integrins was used for detection of the protein expression levels by Western blot (B), as described under Materials and Methods. The results of three experiments are shown; bars represent the mean ± S.D. *, p < 0.05 versus controls after 96 h of incubation (ANOVA followed by a post hoc Dunnett's t test).

Effect of Caspase-8-Specific Inhibitor on Ofloxacin-Stimulated Apoptosis and ₁-Integrin Expression. Stupack et al. (2001) had suggested that "integrin-mediated death" was induced by the activation of caspase-8 in a death receptor-independent manner. Therefore, we further investigated the effect of caspase-8-specific inhibitor z-IETD-fmk on ofloxacin-induced apoptosis. Before incubation with 10 µg/ml ofloxacin for 96 h, the microencapsulated chondrocytes were preincubated for 4 h in the presence or absence of z-IETD-fmk. The percentages of apoptosis are shown in Fig. 12A. The caspase-8 inhibitor caused a concentration-dependent reduction of ofloxacin-induced apoptosis, which was more marked at the concentration of 60 µM. Ofloxacin-stimulated apoptosis increased by approximately 9.5-fold in the absence of these inhibitors but did not significantly increase in the presence of z-IETD-fmk (60 µM). It is noteworthy that the effects of caspase-8 inhibitor were specific because the vehicle dimethyl sulfoxide, used at a similar concentration, had no effect on ofloxacin-stimulated apoptosis. These results indicated that ofloxacin-induced apoptosis was dependent on caspase-8 activation. In addition, we also investigated the effect of caspase-8 inhibition on ofloxacin-stimulated ₁-integrin expression. As shown in Fig. 12B, ₁-integrin markedly declined within 48 h of ofloxacin treatment in the presence of z-IETD-fmk (60 µM), which was similar to the expression mode of ₁-integrin in the presence or absence of z-DEVD-fmk. This suggested that the loss of ₁-integrin was not a direct consequence of caspase-8 activation. Thus, ofloxacin stimulated the activation of caspase-8, inhibited ₁-integrin expression and function, and caused apoptosis in chondrocytes after treatment of 48 h.

View larger version (17K): [in this window] [in a new window]   Fig. 12. Apoptosis induced by ofloxacin involves the activation of caspase-8 in microencapsulated chondrocytes. A, cells were exposed to increasing concentrations (0–60 µM) of caspase-8 inhibitor z-IETD-fmk for 4 h. Ofloxacin (10 µg/ml) was then added, and the cells were incubated for an additional 96 h. The percentage of apoptotic cells was determined by fluorescent staining, as described under Materials and Methods. The values are means ± S.D. of three independent experiments. #, p < 0.05 versus untreated group and *, p < 0.05 versus group of 10 µg/ml ofloxacin alone. B, likewise, cells were treated with 10 µg/ml ofloxacin for up to 96 h after the pretreatment of 60 µM z-DEVD-fmk for 4 h. The antibody against 1-integrin was used for detection of the protein by Western blot, as described under Materials and Methods. The results of three experiments are shown; bars represent the mean ± S.D. *, p < 0.05 versus controls after 96 h of incubation (ANOVA followed by a post hoc Dunnett's t test).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we set out to investigate the possible mechanisms of chondrotoxicity of QNs using the popular drug ofloxacin. An in vitro approach for these studies was used because literally nothing is known about the mechanism of action of these pharmaceuticals and an in vivo assay needs at least some basic mechanistic background. Still, the microencapsulation of isolated chondrocytes allowed us to maintain an almost in vivo like phenotype of the cells for a long time in cell culture compared with monolayer grown chondrocytes and allowed us to use therapeutically relevant concentrations of the compound. Using the microencapsulated culture of juvenile rabbit joint chondrocytes, we have shown that ofloxacin primarily causes apoptosis-mediated cell death in a concentration-dependent manner, a fact not previously reported. We also demonstrated that caspase-3 is involved in ofloxacin-induced apoptotic response of microencapsulated chondrocytes. In addition, we also observed that ofloxacin-induced apoptosis of chondrocytes is associated with alteration of ₁-integrin functions, inhibition on ERK/MAPK signaling pathway, and activation of caspase-8 after exposure of cells to 10 µg/ml ofloxacin for 48 h. Based on our current results, we speculate that ofloxacin-induced apoptosis in chondrocytes results from ₁-integrin disturbance, which is possibly due to alteration of conformational alternation of the extracellular domain in ₁-integrin. Together with previous reports, our current data add further evidence to the hypothesis that a change of integrin function is the primary event causing subsequent changes in the ERK/MAPK signaling pathway, resulting in caspase-8-dependent apoptosis. Herein, some of our observations are unique, and some others corroborate previous studies. We will highlight the outcomes of our study and compare and contrast these with previous studies.

Accumulating evidence has shown that chondrocytes cultured in monolayer in vitro show decreased gene expression of cartilage-specific proteins, such as type II collagen and aggrecan, and quickly dedifferentiate to a more fibroblastic phenotype (van Susante et al., 1995; Enobakhare et al., 1996). In contrast, chondrocytes cultured in alginate gels maintain their differentiated phenotype (round shape) and produce more proteoglycans and type II collagen (van Susante et al., 1995). Our current results are consistent with the above findings. Furthermore, our study reveals that viability of microencapsulated chondrocyte is easily influenced by ofloxacin compared with that cultured in monolayer. In addition, a great advantage of the alginate culture system is that cells can be easily isolated by dissolving the gel matrix by chelating compounds, e.g., EDTA or phosphates, thus allowing further investigations of isolated chondrocytes (Häuselmann et al., 1992). In articular cartilage, chondrocytes receive ECM signals through many receptors, including the hyaluronic acid receptor CD44, annexin V, and integrins (Kurtis et al., 2001). For alginate, however, there is no specific interaction between mammalian cells and the polysaccharide. Furthermore, alginate carries a negative charge balance, such that proteins are not readily adsorbed, due to electrostatic repulsion (Rowley et al., 1999). Thus, we used alginate-microencapsulated chondrocyte to further study the chondrotoxicity of ofloxacin.

We find that ofloxacin-induced cell death in microencapsulated chondrocytes is ascribed to the occurrence of apoptosis, rather than necrosis, which had not been previously reported. The viable fractions of microencapsulated chondrocytes exposed to 10 µg/ml ofloxacin for 72 and 96 h (Fig. 2B) corresponded well to the fractions of dead, apoptotic, and necrotic cells (Fig. 3) determined by fluorescent dye staining. Given the fact that membrane integrity is lost only very late in apoptosis, our data demonstrate that the correlation among apoptotic, dead, and necrotic cells is best obtained by comparing these parameters in the same cell population, thus eliminating the suppressive effects of ofloxacin on cell proliferation.

Proteases of the caspase family are the most important components of apoptotic pathway, and caspase-3 synthesized as an inactive precursor is an early marker and an irreversible point in the progression of apoptosis. Our study showed that activated caspase-3 was involved in ofloxacin-induced apoptotic response of microencapsulated chondrocytes. We also observed the formation of DNA ladder, which is the typical feature of cell apoptosis, and proteolysis of PARP from 116 to 85 kDa, the symbol of caspase activation in the microencapsulated chondrocytes exposed to ofloxacin. These results further confirmed the cell apoptosis-inducing effect of ofloxacin.

It has been shown that QNs seem to disrupt the cation-dependent function of ₁-integrin due to its characteristics of Mg²⁺ chelation (Lomaestro and Bailie, 1991). However, this disruption of function can also affect the integrin-stimulated signaling transduction pathways (Shakibaei et al., 1993). Of special interest is the ERK/MAPK signaling pathway. For this ubiquitous signaling pathway, previous studies with human chondrocytes have shown that its activation is stimulated via ₁-integrin, and subsequent activation of Grb2 and ERK1/2 in ERK/MAPK signaling pathway is extremely important for cell differentiation and survival, inhibition of which can induce apoptosis (Shakibaei et al., 1999, 2001).

Our current data have shown that expression of ₁-integrin mRNA does not change; however, ₁-integrin receptor protein decreased significantly over a 96-h period in ofloxacin-treated microencapsulated chondrocytes. Previous investigations have also shown the alterations of integrin expression of chondrocytes in immature joint cartilage from rats after QNs treatment or after feeding a magnesium-deficient diet (Förster et al., 1996). However, we have found that tyrosine phosphorylation of intracellular integrin-dependent signaling proteins was not present in chondrocytes treated with ofloxacin for 48 h when the expression of ₁-integrin protein significantly declined. Furthermore, supplement of magnesium also makes the expression of ₁-integrin protein, and occurrence of apoptosis remains unchanged under the same time points and concentrations of ofloxacin. Because integrin transition from a low- to high-affinity ligand-binding state may be a direct consequence of conformational changes, involving no change in surface density of integrin (Sanchez-Mateos P et al., 1996). These findings indicate that the ₁-integrin receptors no longer react with the antibody, because the epitope for the antibody is probably changed or deleted from some ₁-integrin molecules. Thus, it is presumed that the absence of functional Mg²⁺ leads to conformational changes of integrin receptor and then perturbs its regular functions after exposure of 48 h to ofloxacin.

For the ERK/MAPK signaling pathway, expression mode of its signaling protein Grb2 and phospho-ERK1/2 corresponds to that of ₁-integrin after ofloxacin treatment. However, inhibition on ofloxacin-induced apoptosis does not affect phospho-ERK1/2 and ₁-integrin expression mode as shown in that treated by ofloxacin alone. In addition, inhibition of ERK/MAPK signaling pathway stimulates the apoptosis of microencapsulated chondrocytes in a concentration-dependent manner, whereas the ₁-integrin levels exhibit no changes under the same conditions. These results further suggest that changes of ₁-integrin expression can inactivate the ERK/MAPK signaling pathway, resulting in apoptosis.

Stupack et al. (2001) have suggested that "integrin-mediated death" is induced by the cytoplasmic domain of unligated ₁-integrin, resulting in the recruitment of caspase-8 to the cell membrane, where it becomes activated in a death receptor-independent manner. In the present study, our observation has indicated that ofloxacin-induced apoptosis involves the caspase-8 activation and that the loss of ₁-integrin is not a direct consequence of caspase-8 activation. Therefore, it is speculated that ₁-integrin cannot ligate to the corresponding ligand because its regular function is perturbed by ofloxacin-stimulated conformational changes, which lead to accumulation of unligated ₁-integrin, subsequent recruitment of caspase-8, inactivation of ERK/MAPK signaling pathway, and ultimate occurrence of apoptosis after the treatment of 48 h.

The reported therapeutic plasma levels of ofloxacin achieved in cartilage of patients are 4 to 10 µg/ml (Hooper and Wolfson, 1985). In our study, the concentrations (5–10 µg/ml) that induce apoptosis in microencapsulated chondrocytes are therefore similar to those reached in the plasma of patients. However, constant concentrations are utilized to act on the cells continuously under our in vitro experimental conditions, whereas concentrations in vivo fluctuate over time.

In summary, the requirement of magnesium for both the desired antibacterial action and the toxic effect on cartilage could explain why all QNs known so far possess the potential for cartilage toxicity. Because ofloxacin, a typical QNs, markedly causes caspase-8-dependent apoptosis of chondrocytes by affecting the levels of ₁-integrin receptor and ERK/MAPK signaling pathway after exposure to the concentrations similar to those of patient's plasma at 48 h, it is likely that other QNs-induced chondrotoxicities involve the same mechanism(s) as ofloxacin. The apoptosis possibly plays an important role in QNs-induced arthropathy, although the question about the detailed mechanism(s) requires further investigation, e.g., the exact relationship between production of cellular H₂O₂, alteration of ₁-integrin function, and caspase-8 activation in the early phase of ofloxacin-induced chondrotoxicity (12–48 h), since the previous data indicated that ofloxacin-induced arthropathy seems to correspond to an early and fairly specific stimulatory effect on the production of H₂O₂ by immature articular chondrocytes (Thuong-Guyot et al., 1994). However, the present evidence of ofloxacin-induced apoptosis in microencapsulated chondrocyte is important for exploring new approaches during preclinical development and clinical risk evaluation of QNs or for strategies to prevent this unusual adverse effect associated with these valuable antimicrobials.

	Acknowledgements

 
We thank Dr. Jing Zhu for critical reviewing of the manuscript.

	Footnotes

 
This work was supported by China Natural Science Foundation Grant 30500641.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.118224.

ABBREVIATIONS: QNs, quinolone(s); PARP, poly(ADP-ribose) polymerase; ERK1/2, extracellular signal-regulated kinase 1/2; CAS 82419-36-1, ofloxacin; RT, reverse transcription; PCR, polymerase chain reaction; Grb2, growth factor receptor-bound protein 2; MAPK, mitogen-activated protein kinase; 3-D, three-dimensional; AO, acridine orange; EB, ethidium bromide; z, benzyloxycarbonyl; fmk, fluoromethyl ketone; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; OD, optical density; PG, proteoglycan; DMB, 1,9-dimethylmethylene blue assay; ANOVA, analysis of variance; FAK, focal adhesion kinase; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene.

Address correspondence to: Dr. Shuangqing Peng, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, People's Republic of China. E-mail: pengsq{at}hotmail.com

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