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Expression of caspase-activated deoxyribonuclease (CAD) and caspase 3 (CPP32) in the cochlea of cisplatin (CDDP)-treated guinea pigs
Auris Nasus Larynx 30 (2003) 219 /225 www.elsevier.com/locate/anl
Expression of caspase-activated deoxyribonuclease (CAD) and caspase 3 (CPP32) in the cochlea of cisplatin (CDDP)-treated guinea pigs Ken-ichi Watanabe *, Shunta Inai, Ken Jinnouchi, Shunkichi Baba, Toshiaki Yagi Department of Oto-Rhino-Laryngology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan Received 6 July 2002; received in revised form 28 February 2003; accepted 14 March 2003
Abstract Cisplatin, an anti-cancer drug, is known to induce apoptosis. During apoptosis, double-stranded DNA is broken into singlestranded DNA by the action of caspases and caspase activated deoxyribonuclease (CAD). We immunohistochemically examined the cochlea of guinea pigs for signs of the apoptosis after the administration of cisplatin. Cisplatin (10 mg/kg b.w.) was intraperitoneally injected to guinea pigs and 3 days later, the animals were sacrificed by intracardiac perfusion of 4% paraformaldehyde. The temporal bones were then removed and immunohistochemically stained for CAD and caspase 3, using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling method. CAD was observed in the stria vascularis and the spiral ligament. Caspase 3 was also detected in the stria vascularis, the spiral ligament and the supporting cells of the organ of Corti. These findings suggest that apoptosis is involved in the cochlear damage observed in cancer patients treated with cisplatin. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Caspase activated deoxyribonuclease (CAD); Caspase 3 (CPP32); Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL); Apoptosis; Cisplatin (CDDP); Cochlea of the ear; Cochleotoxicity
1. Introduction Recently, the biochemical events involved in cell injury have been discussed and it has been demonstrated that apoptosis is an important mechanism of cell death [1 /3]. Apoptosis requires a variety of gene transcriptions and enzyme synthesis. Apoptosis is morphologically characterized by cell shrinkage, chromatin condensation and fragmentation of double-stranded DNA. These events reflect the biochemical reactions that are regulated at distinct steps by proteases. Among those proteases, caspase is known to be activated during apoptosis [4,5]. Liu et al. [6] reported that a caspase inhibitor reduced the number of fragmented nuclei in the explants of auditory sensory cells. Activated caspase 3 (CPP32) cleaves the inhibitor of caspase activated
* Corresponding author. Present address: Department of OtoRhino-Laryngology, Nippon Medical School, Kosugi-cho 1-396, Nakahara-ku, Kawasaki, Kanagawa 211-8533, Japan. Tel.: /81-44733-5181; fax: /81-44-711-8565. E-mail address: [email protected] (K.-i. Watanabe).
deoxyribonuclease (CAD). Then, CAD, which becomes free from the inhibitor of CAD, functions as a nuclease [7,8]. Cisplatin (CDDP), one of platinum-derived anticancer drugs, is now widely used for the treatment of cancer. However, CDDP has some severe side effects, such as nephrotoxicity, myelosuppression and ototoxicity [9]. These side effects are usually the reasons to interrupt chemotherapy. The ototoxicity caused by CDDP is believed to be mediated by free radicals, lipid peroxidation and apoptosis. CDDP generates reactive oxygen species that are also known to activate the apoptotic cascade [10 /13]. We have reported that CDDP activates the expression of inducible nitric oxide synthase in the cochlea [14] and induces the fragmentation of DNA in the cochlea [15]. However, the mechanisms of apoptosis are not fully understood. Thus, we evaluated the influence of CDDP on the cochlea immunohistochemically and electrophysiologically using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method and specific antibodies for CAD or CPP32.
0385-8146/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0385-8146(03)00049-X
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2. Materials and methods 2.1. Materials Twelve guinea pigs weighing between 250 and 350 g were used in this study. All animals were confirmed to have a positive Preyer’s reflex. Animals were anesthetized with 5% (w/v) ketamine hydrochloride (50 mg/kg b.w.) and 2% (w/v) xylazine hydrochloride (10 mg/kg b.w.) before all procedures. The animals were divided into a CDDP group and a control group (NaCl 0.9% (w/ v)). In the CDDP group (n /6), 10 mg/kg b.w. of CDDP (0.5 mg/ml, Bristol /Meyers Squibb K.K., Tokyo, Japan) dissolved in physiological saline (NaCl 0.9% (w/v)) was injected intraperitoneally. In the control group (n / 6), only physiological saline (10 ml/kg, NaCl 0.9% (w/v)) was injected. The kidney in the CDDP group was used as the positive control of TUNEL method, CAD and CPP32. This investigation protocol was in accordance with the guidelines for research involving animals and was approved by the ethical committee of our institution (No. 12 /84 and 13/30). 2.2. TUNEL method The TUNEL method was performed using a commercially available apoptosis in situ detection kit (Wako, No.295-53501, Osaka, Japan). After deparaffinization, the tissue sections were immersed in the protein digestion enzyme for 5 min at 37 8C then in the terminal deoxynucleotidyl transferase solution for 10 min at 37 8C. Subsequently, they were incubated in 3% H2O2 for 5 min, and in peroxidase-conjugated antibody for 10 min at 37 8C. The sections were developed DAB and DAB enhancer for 5 min. The number of the TUNELpositive cells was examined by the semi-quantification method. The darkness of the immunostainings for TUNEL method was graded from 1: no staining to 5: completely dark. We regarded 4 and 5 as the positive staining for TUNEL method. Two doctors independently observed the tissues and counted the number of the TUNEL-positive cells in the tissues. The average numbers were applied in this study. 2.3. Immunohistochemical examination Three days after the i.p. injection of CDDP, all animals were sacrificed. The tissues were fixed through cardiac perfusion with 4% paraformaldehyde (pH 7.4) after flushing out red blood cells with phosphate buffer solution. The both sides of the temporal bones were removed and the cochleas were immersed in the same fixative overnight. The specimens were embedded in paraffin after decalcification with 10% EDTA solution in Tris buffer at pH 7.0 for 5 days.
Each specimen was sectioned into slices 8 mm in thickness using a microtome (Yamato-koki, Tokyo). After removing paraffin, the sections were immersed in 3% H2O2 for 30 min, then in 0.25% Triton X for 10 min. Subsequently, they were incubated with the primary antibody to CPP32 at a 1:2000 dilution (rabbit polyclonal antibody, AF-605-NA, Lot. No. CFZ12, R&D Systems, Inc., Minneapolis, Minn) or CAD at a 1:1000 dilution (rabbit polyclonal antibody, IMG-117, Lot. No. ZB11-1611-JJ04717, IMGENEX, San Diego, CA) overnight. After rinsing the sections with 0.1% Tris buffer solution and normal goat serum, the tissues were incubated with the second antibody at a 1:400 dilution (antirabbit, Dako, Glostrup, Denmark). Processing was ultimately performed with horseradish peroxidase at a 1:100 dilution for 1 h and nickel-enhanced DAB (Wako, Osaka, Japan). 2.4. Auditory brain stem response (ABR) measurement ABR recordings were obtained prior to and 3 days after the injection of each solutions by means of an electrodiagnostic system (COMTEC, Tokyo, Japan). The responses were recorded in the far-field technique. The active electrode was inserted subcutaneously into the ipsilateral pinna, the reference electrode into the contralateral pinna and the ground electrode into the top of the head, respectively. Acoustic stimuli were delivered by an earphone through a small tube inserted into the external ear meatus in a sound proof box. The stimuli consisted of click. They were presented at a rate of 11.1 per s and a duration of 0.11 ms. Responses were accumulated 200 times. The levels of stimuli were lowered from 103 to 33 dBSPL by 5 dB steps. The ABR threshold was determined as the minimum sound level giving the reproducible waveforms. The recordings were repeated twice at the threshold level and the reproducibility was confirmed.
3. Results 3.1. TUNEL method The apoptotic cells were observed in the CDDP treated kidney (Fig. 1a). Fragments of DNA were not detected in all turns of cochlea of normal group (Fig. 1b). In the lateral wall of the cochlea, the nuclei of the stria vascularis were positively stained by the TUNEL method in all turns of cochlea (Fig. 1c/e). There were no signs of fragments of DNA in the spiral ligaments of all turns. In the organ of Corti, there were no apoptotic cells either (Fig. 1c, e). The number of the cells which have positive stainings for TUNEL method is shown in the Fig. 2. In the CDDP group, the fragments of DNA was detected in the stria vascularis of all turns of
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Fig. 1. Paraffin 8 mm-thick sections of the cochlea stained using the TUNEL method 3 days after the injection of CDDP. (a) Positive control; the nuclei of the CDDP treated kidney exhibits a sign of apoptosis (arrow head), /150. (b) Control group; the stria vascularis of the second turn in the control group is shown. No signs of apoptosis can be observed, /150. (c) CDDP group; the fragments of DNA are detected in the stria vascularis, /6. (d) CDDP group; the stria vascularis of the second turn is shown. The nuclei of the stria vascularis (arrow) exhibit signs of apoptosis, /150. (e) CDDP group; in the organ of Corti of basal turn, no apoptotic cells are seen, /150.
cochlea. The number of cells in which the fragments of DNA were detected, tended to be more in the basal turn than in the apex turn, however, these phenomenon were not significant. 3.2. Immunohistochemical expression of CPP32 and CAD The CDDP treated kidney showed a positive immunoreactivity for CPP32 (Fig. 3a) or CAD (Fig. 4a). CPP32 or CAD-staining was not observed in the stria vascularis and the spiral ligament of the control group of all turns of cochlea (Fig. 3b, Fig. 4b). In the CDDP
group, CPP32 and CAD were observed in the lateral wall, especially in the stria vascularis and the spiral ligament (Fig. 3c, d, Fig. 4c, d). These immunoreactivities were detected in all turns of cochlea. In the organ of Corti, CPP32 was detected in the supporting cells (Fig. 3c, e), whereas CAD was not detected (Fig. 4c, e).
3.3. Threshold shifts of ABR The threshold shifts of the ABR before and 3 days after the injection are shown in Table 1. The threshold of the ABR was elevated significantly after 3 days in the
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Fig. 2. The number of the TUNEL-positive cells. In the CDDP group, the fragments of DNA were detected in the stria vascularis of all turns of cochlea. The number of cells in which the fragments of DNA were detected, tended to be more in the basal turn than in the apex turn, however, these phenomenon were not significant.
CDDP group (paired-t, P B/0.05*). In the control group, the threshold shift was not apparent.
4. Discussion It has been shown that apoptosis occurs in the cochlea after the administration of CDDP [1,15]. Apoptosis is controlled by a molecular pathway, which is regulated by proteases. Among proteases, a family of caspases is known to transmit apoptotic signals [4,5,16]. The overexpression of CPP32 also induces apoptosis [4]. On the other hand, CAD, which works as a nuclease, consists of inactive CAD and its inhibitor (ICAD) under physiological condition [7,8]. CPP32 cleaves ICAD, then, the double stranded DNA is broken down fragmented by CAD. We demonstrated that CDDP induces the fragmentation of DNA in the cochlea by in situ detection using the TUNEL method and the hearing disturbance using ABR in this study. CPP32 and CAD were also immunohistochemically detected in the cochlea 3 days after the injection of CDDP. Our results reflected these molecular mechanisms of apoptosis. Another form of cell death, necrosis is reported to cause the fragments of DNA under some conditions, but induce neither the expression of CPP32 nor CAD [17]. It was presumed that CDDP induced the fragments of DNA via apoptotic pathway because of the existence of both CPP32 and CAD. We could detect CPP32, CAD and the fragments of DNA in the stria vascularis, whereas only CPP32 was expressed in the supporting cells of the organ of Corti. These discrepant expressions of proteases are due to the different stages of apoptosis. The death signals activate CPP32 at first, the CAD is activated and the DNA is finally broken into the fragments. The process of
apoptosis was in the final sequence in the stria vascularis, however, not in the supporting cells of the organ of Corti. CDDP is carried to inner ear via blood vessels and might induce cell damage. Komune et al. [18] reported that the ion transport mechanism in the stria vascularis suffered the cytotoxic effects of CDDP. Kohn et al. [19] also reported that CDDP injured the endothelial cells of the stria vascularis. Clinically, it is known that the hearing disturbance caused by CDDP starts especially in high frequencies [9]. The fragments of DNA were detected more in the basal turn than the apex turn. Regardless of the individual differences, this tendency is not contradictory to previous reports. CDDP reacts with DNA directly. Cancer cells are directly destroyed by CDDP. However, normal cells, which consist the structure of the inner ear, do not proliferate. Recently, other pathways, such as free radicals and alterations of calcium currents [20], have been shown to participate in the cytotoxicity of CDDP. We have also reported that the injection of CDDP leads the expression of inducible nitric oxide synthase in the stria vascularis and the supporting cells of the organ of Corti [14]. The expression of inducible nitric oxide synthase induces the apoptotic pathway, which was observed in this study, including the fragmentation of DNA, expression of both CPP32 and CAD. As for the morphological changes caused by CDDP, it is reported that the stria vascularis and the sensory cells show signs of involution [21,22]. Alam et al. [1] reported that the fragments of DNA were detected in the sensory cells after the injection of CDDP. In this study, we detected no apoptotic changes in the organ of Corti. This difference might be due to the different experimental design, that is, the different via of admin-
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Fig. 3. Paraffin 8 mm-thick sections of the cochlea immunohistochemically stained with anti-CPP32 antibody, 3 days after the injection of CDDP. (a) Positive control, anti-CPP32 antibody; the CDDP treated kidney exhibits an intense immunoreactivity for CPP32, /150. (b) Control group, antiCPP32 antibody; the lateral wall of the cochlea in the second turn is shown. No immunoreactivity for CPP32 was detected, /150. (c) CDDP group, anti-CPP32 antibody; CPP32 is seen in the stria vascularis and the spiral ligament, /6. (d) CDDP group, anti-CPP32 antibody; the basal turn of cochlea is shown. CPP32 is seen in the stria vascularis (arrow) and the spiral ligament (arrow head), /150. (e) CDDP group, anti-CPP32 antibody; the second turn of cochlea is shown. Positive immunostaining for CPP32 was detected in the supporting cells of the organ of Corti (arrow), /150.
istration of CDDP, single injection versus multiple injections, the different dose of CDDP and the different species of animals. We also detected CPP32 in the organ of Corti. This means that the apoptotic process was underway in the sensory cells, however, the damage of the sensory cells was irreversible 3 days after the injection of CDDP. Clinical studies have indicated that cochlear damage becomes apparent a few days after the injection of CDDP [21,23]. The incidence of the hearing disturbance is highest in patients, who received a single-high dose injection of CDDP [23,24]. A multiple-low dose injec-
tion also cases the hearing disturbance after cumulative injections. The dose of CDDP is near to LD50 in this study. This dose is higher than the clinical use. In the field of the head and neck surgery, the clinical application of CDDP is around 0.2 /0.6 mg/kg [23] for 5 /7 days and the dose of over 2 mg/kg of CDDP is reported to be risky to cause the hearing disturbance [23]. Even in the experiments for chronic ototoxicity of CDDP, the dose of CDDP is reported to be around 1 mg/kg for several days, which is still higher than the clinical dose [25,26]. We selected a high and single dose of CDDP in this study to ensure the inner ear damage for the model of
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Acknowledgements We thank Ms. Sachiko Saito and Mr. Kensuke Watanabe for their technical assistance. This investigation was supported by a grant from the ministry of Education, Culture, Sports, Science and Technology (No. 659-7053-12770997).
References
Fig. 4. Paraffin 8 mm-thick sections of the cochlea immunohistochemically stained with anti-CAD antibody, 3 days after the injection of CDDP. (a) Positive control, anti-CAD antibody; the CDDP treated kidney exhibits an intense immunoreactivity for CAD, /150. (b) Control group, anti-CAD antibody; the lateral wall of the cochlea in the second turn is shown. No immunoreactivity for CAD was detected, /150. (c) CDDP group, anti-CAD antibody; the lateral wall of the cochlea exhibits positive staining for CAD, /6. (d) CDDP group, anti-CAD antibody; the second turn of cochlea is shown. CAD is seen in the stria vascularis (arrow) and the spiral ligament (arrow head), / 150. (e) CDDP group, anti-CAD antibody; the organ of Corti in the second turn is shown. No immunostaining for CAD is seen, /150.
Table 1 Changes of the threshold level of ABR
acute ototoxicity of CDDP. However, it is difficult to say which model, that is, a high-single dose or lowmultiple dose, is appropriate to extrapolate it into the human. In conclusion, the systemic injection of CDDP induced the expression of CAD and CPP32 in the cochlea. The apoptotic cells were mainly observed in the stria vascularis of the cochlea. Our study indicates that apoptosis is involved in the cochleotoxicity of CDDP.
[1] Alam SA, Ikeda K, Oshima T, Suzuki M, Kawase T, Kikuchi T, et al. Cisplatin-induced apoptotic cell death in Mongolian gerbil cochlea. Hear Res 2000;141:28 /38. [2] Miller LJ, Marx J. Apoptosis. Science 1998;281:1301. [3] Nakagawa T, Yamane H, Takayama M, Sunami K, Nakai Y. Apoptosis of guinea pig cochlear hair cells following chronic aminoglycoside treatment. Eur Arch Otorhinolaryngol 1998;255:127 /31. [4] Patel T, Gores GJ, Kaufmann SH. The role of proteases during apoptosis. FASEB J 1996;588:587 /97. [5] Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998;281:1312 /6. [6] Liu W, Staecker H, Stupak H, Malgrange B, Lefebvre P, Van De Water TR. Caspase inhibitors prevent cisplatin-induced apoptosis of auditory sensory cells. Neuroreport 1998;9:2609 /14. [7] Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata SA. Caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 1998;391:43 /50. [8] Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibition in CAD activation and DNA degradation during apoptosis. Nature 1998;391:96 /9. [9] McKeage MJ. Comparative adverse effect profiles of platinum drugs. Drug Safety 1995;13:228 /44. [10] Kondo S, Barna BP, Morimura T, Takeuchi J, Yuan J, Akbasak A, et al. Interleukin-1b-converting enzyme mediates cisplatininduced apoptosis in malignant glioma cells. Cancer Res 1995;55:6166 /71. [11] Kopke RD, Liu W, Gabaizadeh R, Jacono A, Feghali J, Spray D, et al. Use of organotypic cultures of Corti’s organ to study the protective effects of antioxidant molecules on icsplatin-induced damage of auditory hair cells. Am J Otol 1997;18:559 /71. [12] Maldonado V, Mele´ndez-Zajgla J, Ortega A. Modulation of NFkappaB, p53 and Bcl-2 in apoptosis induced by cisplatin in HeLa cells. Mutat Res 1997;381:67 /75. [13] Ravi R, Somani SM, Rybak LP. Mechanism of cisplatin ototoxicity: antioxidant system. Pharmacol Toxicol 1995;76:386 /94. [14] Watanabe K, Hess A, Michel O, Yagi T. NOS inhibitor reduces the apoptotic change in the cisplatinum (CDDP) treated cochlea of guinea pigs. Anti-Cancer Drugs 2000;11:731 /5. [15] Watanabe K, Hess A, Bloch W, Michel O. NOS-inhibitor blocks ototoxic side effect of CDDP in the guinea pigs. Anti-Cancer Drugs 2000;11:401 /6. [16] Liu X, Zou H, Slaughter C, Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 1997;89:175 /84. [17] Dong Z, Saikumar P, Weinberg JM, Venkatachalam MA. Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death. Am J Pathol 1997;151:1205 / 13. [18] Komune S, Asakuma S, Snow JB. Pathophysiology of the ototoxicity of cis-diaminedichloroplatinum. Otolaryngol Head Neck Surg 1981;89:275 /82.
K.-i. Watanabe et al. / Auris Nasus Larynx 30 (2003) 219 /225 [19] Kohn S, Fradis M, Podoshin L, Ben-David J, Zidan J, Robinson E. Endothelial injury of capillaries in the stria vascularis of guinea pigs treated with cisplatin and gentamicin. Ultrastruct Pathol 1997;21:289 /99. [20] Yamamoto T, Kakehata S, Saito T, Saito H, Akaike N. Cisplatin blocks voltage-dependent calcium current in dissociated outer hair cells of guinea-pig cochlea. Brain Res 1994;648:296 /8. [21] Moroso MJ, Blair RL. A review of cis -platinum ototoxicity. J Otolaryngol 1983;12:365 /9. [22] Tange RA, Vuzevski VD. Changes in the stria vascularis of the guinea pig due to cis-platinum. Arch Otorhinolaryngol 1984;239:41 /7.
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[23] Lippman AJ, Helson C, Helson L, Krakoff IH. Clinical trials of cis-diamminedichloroplatinum (NSC-119875). Cancer Chemother Rep 1973;57:191 /200. [24] Nihonkayaku Co. Ltd.: dose of cisplatin, In: Nihonkayaku CoLtd. (Eds.) randa inj. Tokyo, Nihonkayaku Co.Ltd., 1998. pp 10 /21. [25] Taudy M, Syka J, Popelar J, Ulehlova L. Carboplatin and cisplatin ototoxicity in guinea pigs. Audiology 1992;31:293 / 9. [26] Laurell G, Engstro¨m B. The ototoxic side effect of cisplatin on guinea pigs in relation to dosage. Hear Res 1989;38:27 / 34.
Expression of caspase-activated deoxyribonuclease (CAD) and caspase 3 (CPP32) in the cochlea of cisplatin (CDDP)-treated guinea pigs Ken-ichi Watanabe *, Shunta Inai, Ken Jinnouchi, Shunkichi Baba, Toshiaki Yagi Department of Oto-Rhino-Laryngology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan Received 6 July 2002; received in revised form 28 February 2003; accepted 14 March 2003
Abstract Cisplatin, an anti-cancer drug, is known to induce apoptosis. During apoptosis, double-stranded DNA is broken into singlestranded DNA by the action of caspases and caspase activated deoxyribonuclease (CAD). We immunohistochemically examined the cochlea of guinea pigs for signs of the apoptosis after the administration of cisplatin. Cisplatin (10 mg/kg b.w.) was intraperitoneally injected to guinea pigs and 3 days later, the animals were sacrificed by intracardiac perfusion of 4% paraformaldehyde. The temporal bones were then removed and immunohistochemically stained for CAD and caspase 3, using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling method. CAD was observed in the stria vascularis and the spiral ligament. Caspase 3 was also detected in the stria vascularis, the spiral ligament and the supporting cells of the organ of Corti. These findings suggest that apoptosis is involved in the cochlear damage observed in cancer patients treated with cisplatin. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Caspase activated deoxyribonuclease (CAD); Caspase 3 (CPP32); Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL); Apoptosis; Cisplatin (CDDP); Cochlea of the ear; Cochleotoxicity
1. Introduction Recently, the biochemical events involved in cell injury have been discussed and it has been demonstrated that apoptosis is an important mechanism of cell death [1 /3]. Apoptosis requires a variety of gene transcriptions and enzyme synthesis. Apoptosis is morphologically characterized by cell shrinkage, chromatin condensation and fragmentation of double-stranded DNA. These events reflect the biochemical reactions that are regulated at distinct steps by proteases. Among those proteases, caspase is known to be activated during apoptosis [4,5]. Liu et al. [6] reported that a caspase inhibitor reduced the number of fragmented nuclei in the explants of auditory sensory cells. Activated caspase 3 (CPP32) cleaves the inhibitor of caspase activated
* Corresponding author. Present address: Department of OtoRhino-Laryngology, Nippon Medical School, Kosugi-cho 1-396, Nakahara-ku, Kawasaki, Kanagawa 211-8533, Japan. Tel.: /81-44733-5181; fax: /81-44-711-8565. E-mail address: [email protected] (K.-i. Watanabe).
deoxyribonuclease (CAD). Then, CAD, which becomes free from the inhibitor of CAD, functions as a nuclease [7,8]. Cisplatin (CDDP), one of platinum-derived anticancer drugs, is now widely used for the treatment of cancer. However, CDDP has some severe side effects, such as nephrotoxicity, myelosuppression and ototoxicity [9]. These side effects are usually the reasons to interrupt chemotherapy. The ototoxicity caused by CDDP is believed to be mediated by free radicals, lipid peroxidation and apoptosis. CDDP generates reactive oxygen species that are also known to activate the apoptotic cascade [10 /13]. We have reported that CDDP activates the expression of inducible nitric oxide synthase in the cochlea [14] and induces the fragmentation of DNA in the cochlea [15]. However, the mechanisms of apoptosis are not fully understood. Thus, we evaluated the influence of CDDP on the cochlea immunohistochemically and electrophysiologically using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method and specific antibodies for CAD or CPP32.
0385-8146/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0385-8146(03)00049-X
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2. Materials and methods 2.1. Materials Twelve guinea pigs weighing between 250 and 350 g were used in this study. All animals were confirmed to have a positive Preyer’s reflex. Animals were anesthetized with 5% (w/v) ketamine hydrochloride (50 mg/kg b.w.) and 2% (w/v) xylazine hydrochloride (10 mg/kg b.w.) before all procedures. The animals were divided into a CDDP group and a control group (NaCl 0.9% (w/ v)). In the CDDP group (n /6), 10 mg/kg b.w. of CDDP (0.5 mg/ml, Bristol /Meyers Squibb K.K., Tokyo, Japan) dissolved in physiological saline (NaCl 0.9% (w/v)) was injected intraperitoneally. In the control group (n / 6), only physiological saline (10 ml/kg, NaCl 0.9% (w/v)) was injected. The kidney in the CDDP group was used as the positive control of TUNEL method, CAD and CPP32. This investigation protocol was in accordance with the guidelines for research involving animals and was approved by the ethical committee of our institution (No. 12 /84 and 13/30). 2.2. TUNEL method The TUNEL method was performed using a commercially available apoptosis in situ detection kit (Wako, No.295-53501, Osaka, Japan). After deparaffinization, the tissue sections were immersed in the protein digestion enzyme for 5 min at 37 8C then in the terminal deoxynucleotidyl transferase solution for 10 min at 37 8C. Subsequently, they were incubated in 3% H2O2 for 5 min, and in peroxidase-conjugated antibody for 10 min at 37 8C. The sections were developed DAB and DAB enhancer for 5 min. The number of the TUNELpositive cells was examined by the semi-quantification method. The darkness of the immunostainings for TUNEL method was graded from 1: no staining to 5: completely dark. We regarded 4 and 5 as the positive staining for TUNEL method. Two doctors independently observed the tissues and counted the number of the TUNEL-positive cells in the tissues. The average numbers were applied in this study. 2.3. Immunohistochemical examination Three days after the i.p. injection of CDDP, all animals were sacrificed. The tissues were fixed through cardiac perfusion with 4% paraformaldehyde (pH 7.4) after flushing out red blood cells with phosphate buffer solution. The both sides of the temporal bones were removed and the cochleas were immersed in the same fixative overnight. The specimens were embedded in paraffin after decalcification with 10% EDTA solution in Tris buffer at pH 7.0 for 5 days.
Each specimen was sectioned into slices 8 mm in thickness using a microtome (Yamato-koki, Tokyo). After removing paraffin, the sections were immersed in 3% H2O2 for 30 min, then in 0.25% Triton X for 10 min. Subsequently, they were incubated with the primary antibody to CPP32 at a 1:2000 dilution (rabbit polyclonal antibody, AF-605-NA, Lot. No. CFZ12, R&D Systems, Inc., Minneapolis, Minn) or CAD at a 1:1000 dilution (rabbit polyclonal antibody, IMG-117, Lot. No. ZB11-1611-JJ04717, IMGENEX, San Diego, CA) overnight. After rinsing the sections with 0.1% Tris buffer solution and normal goat serum, the tissues were incubated with the second antibody at a 1:400 dilution (antirabbit, Dako, Glostrup, Denmark). Processing was ultimately performed with horseradish peroxidase at a 1:100 dilution for 1 h and nickel-enhanced DAB (Wako, Osaka, Japan). 2.4. Auditory brain stem response (ABR) measurement ABR recordings were obtained prior to and 3 days after the injection of each solutions by means of an electrodiagnostic system (COMTEC, Tokyo, Japan). The responses were recorded in the far-field technique. The active electrode was inserted subcutaneously into the ipsilateral pinna, the reference electrode into the contralateral pinna and the ground electrode into the top of the head, respectively. Acoustic stimuli were delivered by an earphone through a small tube inserted into the external ear meatus in a sound proof box. The stimuli consisted of click. They were presented at a rate of 11.1 per s and a duration of 0.11 ms. Responses were accumulated 200 times. The levels of stimuli were lowered from 103 to 33 dBSPL by 5 dB steps. The ABR threshold was determined as the minimum sound level giving the reproducible waveforms. The recordings were repeated twice at the threshold level and the reproducibility was confirmed.
3. Results 3.1. TUNEL method The apoptotic cells were observed in the CDDP treated kidney (Fig. 1a). Fragments of DNA were not detected in all turns of cochlea of normal group (Fig. 1b). In the lateral wall of the cochlea, the nuclei of the stria vascularis were positively stained by the TUNEL method in all turns of cochlea (Fig. 1c/e). There were no signs of fragments of DNA in the spiral ligaments of all turns. In the organ of Corti, there were no apoptotic cells either (Fig. 1c, e). The number of the cells which have positive stainings for TUNEL method is shown in the Fig. 2. In the CDDP group, the fragments of DNA was detected in the stria vascularis of all turns of
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Fig. 1. Paraffin 8 mm-thick sections of the cochlea stained using the TUNEL method 3 days after the injection of CDDP. (a) Positive control; the nuclei of the CDDP treated kidney exhibits a sign of apoptosis (arrow head), /150. (b) Control group; the stria vascularis of the second turn in the control group is shown. No signs of apoptosis can be observed, /150. (c) CDDP group; the fragments of DNA are detected in the stria vascularis, /6. (d) CDDP group; the stria vascularis of the second turn is shown. The nuclei of the stria vascularis (arrow) exhibit signs of apoptosis, /150. (e) CDDP group; in the organ of Corti of basal turn, no apoptotic cells are seen, /150.
cochlea. The number of cells in which the fragments of DNA were detected, tended to be more in the basal turn than in the apex turn, however, these phenomenon were not significant. 3.2. Immunohistochemical expression of CPP32 and CAD The CDDP treated kidney showed a positive immunoreactivity for CPP32 (Fig. 3a) or CAD (Fig. 4a). CPP32 or CAD-staining was not observed in the stria vascularis and the spiral ligament of the control group of all turns of cochlea (Fig. 3b, Fig. 4b). In the CDDP
group, CPP32 and CAD were observed in the lateral wall, especially in the stria vascularis and the spiral ligament (Fig. 3c, d, Fig. 4c, d). These immunoreactivities were detected in all turns of cochlea. In the organ of Corti, CPP32 was detected in the supporting cells (Fig. 3c, e), whereas CAD was not detected (Fig. 4c, e).
3.3. Threshold shifts of ABR The threshold shifts of the ABR before and 3 days after the injection are shown in Table 1. The threshold of the ABR was elevated significantly after 3 days in the
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Fig. 2. The number of the TUNEL-positive cells. In the CDDP group, the fragments of DNA were detected in the stria vascularis of all turns of cochlea. The number of cells in which the fragments of DNA were detected, tended to be more in the basal turn than in the apex turn, however, these phenomenon were not significant.
CDDP group (paired-t, P B/0.05*). In the control group, the threshold shift was not apparent.
4. Discussion It has been shown that apoptosis occurs in the cochlea after the administration of CDDP [1,15]. Apoptosis is controlled by a molecular pathway, which is regulated by proteases. Among proteases, a family of caspases is known to transmit apoptotic signals [4,5,16]. The overexpression of CPP32 also induces apoptosis [4]. On the other hand, CAD, which works as a nuclease, consists of inactive CAD and its inhibitor (ICAD) under physiological condition [7,8]. CPP32 cleaves ICAD, then, the double stranded DNA is broken down fragmented by CAD. We demonstrated that CDDP induces the fragmentation of DNA in the cochlea by in situ detection using the TUNEL method and the hearing disturbance using ABR in this study. CPP32 and CAD were also immunohistochemically detected in the cochlea 3 days after the injection of CDDP. Our results reflected these molecular mechanisms of apoptosis. Another form of cell death, necrosis is reported to cause the fragments of DNA under some conditions, but induce neither the expression of CPP32 nor CAD [17]. It was presumed that CDDP induced the fragments of DNA via apoptotic pathway because of the existence of both CPP32 and CAD. We could detect CPP32, CAD and the fragments of DNA in the stria vascularis, whereas only CPP32 was expressed in the supporting cells of the organ of Corti. These discrepant expressions of proteases are due to the different stages of apoptosis. The death signals activate CPP32 at first, the CAD is activated and the DNA is finally broken into the fragments. The process of
apoptosis was in the final sequence in the stria vascularis, however, not in the supporting cells of the organ of Corti. CDDP is carried to inner ear via blood vessels and might induce cell damage. Komune et al. [18] reported that the ion transport mechanism in the stria vascularis suffered the cytotoxic effects of CDDP. Kohn et al. [19] also reported that CDDP injured the endothelial cells of the stria vascularis. Clinically, it is known that the hearing disturbance caused by CDDP starts especially in high frequencies [9]. The fragments of DNA were detected more in the basal turn than the apex turn. Regardless of the individual differences, this tendency is not contradictory to previous reports. CDDP reacts with DNA directly. Cancer cells are directly destroyed by CDDP. However, normal cells, which consist the structure of the inner ear, do not proliferate. Recently, other pathways, such as free radicals and alterations of calcium currents [20], have been shown to participate in the cytotoxicity of CDDP. We have also reported that the injection of CDDP leads the expression of inducible nitric oxide synthase in the stria vascularis and the supporting cells of the organ of Corti [14]. The expression of inducible nitric oxide synthase induces the apoptotic pathway, which was observed in this study, including the fragmentation of DNA, expression of both CPP32 and CAD. As for the morphological changes caused by CDDP, it is reported that the stria vascularis and the sensory cells show signs of involution [21,22]. Alam et al. [1] reported that the fragments of DNA were detected in the sensory cells after the injection of CDDP. In this study, we detected no apoptotic changes in the organ of Corti. This difference might be due to the different experimental design, that is, the different via of admin-
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Fig. 3. Paraffin 8 mm-thick sections of the cochlea immunohistochemically stained with anti-CPP32 antibody, 3 days after the injection of CDDP. (a) Positive control, anti-CPP32 antibody; the CDDP treated kidney exhibits an intense immunoreactivity for CPP32, /150. (b) Control group, antiCPP32 antibody; the lateral wall of the cochlea in the second turn is shown. No immunoreactivity for CPP32 was detected, /150. (c) CDDP group, anti-CPP32 antibody; CPP32 is seen in the stria vascularis and the spiral ligament, /6. (d) CDDP group, anti-CPP32 antibody; the basal turn of cochlea is shown. CPP32 is seen in the stria vascularis (arrow) and the spiral ligament (arrow head), /150. (e) CDDP group, anti-CPP32 antibody; the second turn of cochlea is shown. Positive immunostaining for CPP32 was detected in the supporting cells of the organ of Corti (arrow), /150.
istration of CDDP, single injection versus multiple injections, the different dose of CDDP and the different species of animals. We also detected CPP32 in the organ of Corti. This means that the apoptotic process was underway in the sensory cells, however, the damage of the sensory cells was irreversible 3 days after the injection of CDDP. Clinical studies have indicated that cochlear damage becomes apparent a few days after the injection of CDDP [21,23]. The incidence of the hearing disturbance is highest in patients, who received a single-high dose injection of CDDP [23,24]. A multiple-low dose injec-
tion also cases the hearing disturbance after cumulative injections. The dose of CDDP is near to LD50 in this study. This dose is higher than the clinical use. In the field of the head and neck surgery, the clinical application of CDDP is around 0.2 /0.6 mg/kg [23] for 5 /7 days and the dose of over 2 mg/kg of CDDP is reported to be risky to cause the hearing disturbance [23]. Even in the experiments for chronic ototoxicity of CDDP, the dose of CDDP is reported to be around 1 mg/kg for several days, which is still higher than the clinical dose [25,26]. We selected a high and single dose of CDDP in this study to ensure the inner ear damage for the model of
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Acknowledgements We thank Ms. Sachiko Saito and Mr. Kensuke Watanabe for their technical assistance. This investigation was supported by a grant from the ministry of Education, Culture, Sports, Science and Technology (No. 659-7053-12770997).
References
Fig. 4. Paraffin 8 mm-thick sections of the cochlea immunohistochemically stained with anti-CAD antibody, 3 days after the injection of CDDP. (a) Positive control, anti-CAD antibody; the CDDP treated kidney exhibits an intense immunoreactivity for CAD, /150. (b) Control group, anti-CAD antibody; the lateral wall of the cochlea in the second turn is shown. No immunoreactivity for CAD was detected, /150. (c) CDDP group, anti-CAD antibody; the lateral wall of the cochlea exhibits positive staining for CAD, /6. (d) CDDP group, anti-CAD antibody; the second turn of cochlea is shown. CAD is seen in the stria vascularis (arrow) and the spiral ligament (arrow head), / 150. (e) CDDP group, anti-CAD antibody; the organ of Corti in the second turn is shown. No immunostaining for CAD is seen, /150.
Table 1 Changes of the threshold level of ABR
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