Aminoglycoside antibiotics, loop diuretics, antineoplastic agents and other commonly used pharmacological drugs are ototoxic. Understanding of the cellular and molecular mechanisms underlying drug ototoxicity, however, has been hampered by the limited availability of inner ear tissues and drug side effects on laboratory animals. Immortalized cell lines derived from the auditory sensory organ, sensitive to ototoxic drugs and growing in environments that can be systematically manipulated, would facilitate the research directed at elucidating these mechanisms. Such immortalized cell lines could also be used to discover novel therapeutic agents for preventing drug-induced sensorineural hearing loss. Here, we report a conditionally immortalized organ of Corti-derived epithelial cell line, which shows evidence of activation of apoptosis when exposed to known ototoxic drugs. This cell line may be an excellent in vitro system to investigate the cellular and molecular mechanisms involved in ototoxicity and for screening of the potential ototoxicity or otoprotective properties of new pharmacological drugs.

Keywords:
Cell line, Cochlea, Ototoxic drugs, Aminoglycoside antibiotics, Drug screening, Immortomouse
1.
Anderson RG, Pathak RK: Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments. Cell 1985;40:635–643.
2.
Barald KF, Lindberg KH, Hardiman K, Kavka AI, Lewis JE, Victor JC, Gardner CA, Poniatowski A: Immortalized cell lines from embryonic avian and murine otocysts: Tools for molecular studies of the developing inner ear. Int J Dev Neurosci 1997;15:523–540.
3.
Belyantseva IA, Adler HJ, Curi R, Frolenkov GI, Kachar B: Expression and localization of prestin and the sugar transporter GLUT-5 during development of electromotility in cochlear outer hair cells. J Neurosci 2000;20:RC116.
4.
Bermingham NA, Hassan BA, Price SD, Vollrath MA, Ben-Arie N, Eatock RA, Bellen HJ, Lysakowski A, Zoghbi HY: Math1: An essential gene for the generation of inner ear hair cells. Science 1999;284:1837–1841.
5.
Bertolaso L, Martini A, Bindini D, Lanzoni I, Parmeggiani A, Vitali C, Kalinec G, Kalinec F, Capitani S, Previati M: Apoptosis in the OC-k3 immortalized cell line treated with different agents. Audiology 2001;40:327–335.
6.
Boëda B, Weil D, Petit C: A specific promoter of the sensory cells of the inner ear defined by transgenesis. Hum Mol Genet 2001;10:1581–1589.
7.
Chen H, Thalmann I, Adams JC, Avraham KB, Copeland NG, Jenkins NA, Beier DR, Corey DP, Thalmann R, Duyk GM: cDNA cloning, tissue distribution, and chromosomal localization of Ocp2, a gene encoding a putative transcription-associated factor predominantly expressed in the auditory organs. Genomics 1995;27:389–398.
8.
Chen P, Johnson JE, Zoghbi HY, Segil N: The role of Math1 in inner ear development: Uncoupling the establishment of the sensory primordium from hair cell fate determination. Development 2002;129:2495–2505.
9.
Coleman JK, Jianzhong L, Huang X, Wood K, Jackson R, Hoffer ME, Kopke RD: Acetyl-L-carnitine attenuates noise-induced hearing loss as measured by ABR in the chinchilla. 24th ARO Midwinter Meeting, St. Petersburg Beach, Fla., 2001, p 119a.
10.
Dallos P, Fakler B: Prestin, a new type of motor protein. Nat Rev Mol Cell Biol 2002;3:104–111.
11.
Dechesne CJ, Winsky L, Moniot B, Raymond J: Localization of calretinin mRNA in rat and guinea pig inner ear by in situ hybridization using radioactive and non-radioactive probes. Hear Res 1993;69:91–97.
12.
de Groot JCMJ, Meeuwesen F, Ruizendaal WE, Veldman JE: Ultrastructural localisation of gentamicin in the cochlea. Hear Res 1990;50:35–42.
13.
Devarajan P, Savoca M, Park M-S, Castaneda MP, Esteban N, Kalinec G, Kalinec F: Cisplatin-induced apoptosis of auditory cells: Role of death receptor and mitochondrial pathways. Hear Res 2002;174:45–54.
14.
El Mouedden M, Laurent G, Mingeot-Leclercq M-P, Tulkens PM: Gentamicin-induced apoptosis in renal cell lines and embryonic rat fibroblasts. Toxicol Sci 2000;56:229–239.
15.
Espreafico EM, Coling DE, Tsakraklides V, Krogh K, Wolenski JS, Kalinec G, Kachar B: Localization of myosin-V in the centrosome. Proc Natl Acad Sci USA 1998;95:8636–8641.
16.
Ford DM, Dahl RH, Lamp CA, Molitoris BA: Apically and basolaterally internalized aminoglycosides colocalize in LLC-PK1 lysosomes and alter cell function. Am J Physiol 1994;266:C52–C57.
17.
Forge A, Schacht J: Aminoglycoside antibiotics. Audiol Neurootol 2000;5:3–22.
18.
Giurgea-Marion L, Toubeau G, Laurent G, Heuson-Stiennon JA, Tulkens PM: Impairment of lysosome-pinocytic vesicle fusion in rat kidney proximal tubules after treatment with gentamicin at low doses. Toxicol Appl Pharmacol 1986;86:271–285.
19.
Hasson T, Heintzelman MB, Santos-Sacchi J, Corey DP, Mooseker MS: Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type 1B. Proc Natl Acad Sci USA 1995;92:9815–9819.
20.
Hasson T, Mooseker MS: Vertebrate unconventional myosins. J Biol Chem 1996;271:16431–16434.
21.
Henley CM, Rybak LP: Ototoxicity in developing mammals. Brain Res Brain Res Rev 1995;20:68–90.
22.
Holley MC, Lawlor PW: Production of conditionally immortalised cell lines from a transgenic mouse. Audiol Neurootol 1997;2:25–35.
23.
Huang MY, Schacht J: Drug-induced ototoxicity. Pathogenesis and prevention. Med Toxicol Adverse Drug Exp 1989;4:452–467.
24.
Huang T, Cheng AG, Stupak H, Liu W, Kim A, Staeckert H, Lefebvre PP, Malgrange B, Kopke R, Moonen G, Van De Water T: Oxidative stress-induced apoptosis of cochlear sensory cells: Otoprotective strategies. Int J Dev Neurosci 2000;18:259–270.
25.
Jagger DJ, Griesinger CB, Rivolta MN, Holley MC, Ashmore JF: Calcium signalling mediated by the 9 acetylcholine receptor in a cochlear cell line from the Immortomouse. J Physiol 2000;527:49–54.
26.
Jat PS, Noble MD, Ataliotis P, Tanaka Y, Yannoutsos N, Larsen L, Kioussis D: Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc Natl Acad Sci USA 1991;88:5096–5100.
27.
Jordan P, Carmo-Fonseca M: Molecular mechanisms involved in cisplatin cytotoxicity. Cell Mol Life Sci 2000;57:1229–1235.
28.
Kalinec F, Kalinec G, Boukhvalova M, Kachar B: Establishment and characterization of conditionally immortalized organ of Corti cell lines. Cell Biol Int 1999;23:175–184.
29.
Kalinec F, Kalinec GM, Kachar B: Development of organ of Corti and stria vascularis cell lines from transgenic mice. 19th ARO Midwinter Meeting, St. Petersburg Beach, Fla., 1996, p 577a.
30.
Kalinec GM, Esteban N, Kalinec F: Protective role of L-carnitine on the ototoxic effects of cisplatin in newborn guinea pigs. 25th ARO Midwinter Meeting, St. Petersburg Beach, Fla., 2002, p 855a.
31.
Kelley MW, Xu X-M, Wagner MA, Warchol ME, Corwin JT: The developing organ of Corti contains retinoic acid and forms supernumerary hair cells in response to exogenous retinoic acid in culture. Development 1993;119:1041–1053.
32.
Kotecha B, Richardson GP: Ototoxicity in vitro: Effects of neomycin, gentamicin, dihydrostreptomycin, amikacin, spectinomycin, neamine, spermine and poly-L-Lysine. Hear Res 1994;73:173–184.
33.
Lautermann J, ten Cate W-JF: Postnatal expression of the α-thyroid hormone receptor in the rat cochlea. Hear Res 1997;107:23–28.
34.
Lautermann J, ten Cate W-JF, Altenhoff P, Grummer R, Traub O, Frank H, Jahnke K, Winterhager E: Expression of the gap-junction connexins 26 and 30 in the rat cochlea. Cell Tissue Res 1998;294:415–420.
35.
Lawlor P, Marcotti W, Rivolta MN, Kros CJ, Holley MC: Differentiation of mammalian vestibular hair cells from conditionally immortal, postnatal supporting cells. J Neurosci 1999;19:9445–9458.
36.
Lendahl U, Zimmerman LB, McKay RD: CNS stem cells express a new class of intermediate filament protein. Cell 1990;60:585–595.
37.
Lerner SA, Schmitt BA, Seligsohn R, Matz GJ: Comparative study of ototoxicity and nephrotoxicity in patients randomly assigned to treatment with amikacin or gentamicin. Am J Med 1986;80:98–104.
38.
Löwenheim H, Kil J, Gültig K, Zenner H-P: Determination of hair cell degeneration and hair cell death in neomycin treated cultures of the neonatal rat cochlea. Hear Res 1999;128:16–26.
39.
Malgrange B, Belachew S, Thiry M, Nguyen L, Rogister B, Alvarez ML, Rigo JM, Van De Water TR, Moonen G, Lefebvre PP: Proliferative generation of mammalian auditory hair cells in culture. Mech Dev 2002;112:79–88.
40.
McKay R: Stem cells in the central nervous system. Science 1997;276:66–71.
41.
McKay R, Renfranz P, Cunningham M: Immortalized stem cells from the central nervous system. C R Acad Sci III 1993;316:1452–1457.
42.
Ohkuma S, Poole B: Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci USA 1978;75:3327–3331.
43.
Pack AK, Slepecky NB: Cytoskeletal and calcium-binding proteins in the mammalian organ of Corti: Cell type-specific proteins displaying longitudinal and radial gradients. Hear Res 1995;91:119–135.
44.
Pirvola U, Cao Y, Oellig C, Suoqiang Z, Pettersson RF, Ylikoski J: The site of action of neuronal acidic fibroblast growth factor is the organ of Corti of the rat cochlea. Proc Natl Acad Sci USA 1995;92:9269–9273.
45.
Richardson GP, Forge A, Kros CJ, Fleming J, Brown SD, Steel KP: Myosin VIIA is required for aminoglycoside accumulation in cochlear hair cells. J Neurosci 1997;17:9506–9519.
46.
Richardson GP, Russell IJ: Cochlear cultures as a model system for studying aminoglycoside induced ototoxicity. Hear Res 1991;53:293–311.
47.
Rivolta MN, Grix N, Lawlor P, Ashmore JF, Jagger DJ, Holley MC: Auditory hair cell precursors immortalized from the mammalian inner ear. Proc R Soc Lond B Biol Sci 1998;265:1595–1603.
48.
Ruben RJ: Development of the inner ear of the mouse: A radioautographic study of terminal mitoses. Acta Otolaryngol 1967; (suppl 220):1–44.
49.
Rybak L: Ototoxicity. Curr Opin Otolaryngol Head Neck Surg 1996;4:302–307.
50.
Schecterson L, Bothwell M: Neurotrophin and neurotrophin receptor mRNA expression in developing inner ear. Hear Res 1994;73:92–100.
51.
Seidman M, Khan M, Bain U, Shirwany N, Quirk W: Biologic activity of mitochondrial metabolites on aging and age-related hearing loss. Am J Otol 2000;21:161–167.
52.
Slepecky NB, Ulfendahl M: Evidence for calcium-binding proteins and calcium-dependent regulatory proteins in sensory cells of the organ of Corti. Hear Res 1993;70:73–84.
53.
Stemple DL, Mahanthappa NK: Neural stem cells are blasting off. Neuron 1997;18:1–4.
54.
Stone JS, Leaño SG, Baker LP, Rubel EW: Hair cell differentiation in chick cochlear epithelium after aminoglycoside toxicity: In vivo and in vitro observations. J Neurosci 1996;16:6157–6174.
55.
Tulkens P, Trouet A: The uptake and intracellular accumulation of aminoglycoside antibiotics in lysosomes of cultured rat fibroblasts. Biochem Pharmacol 1978;27:415–424.
56.
Wheeler EF, Bothwell M, Schecterson LC, von Bartheld CS: Expression of BDNF and NT-3 mRNA in hair cells of the organ of Corti: Quantitative analysis in developing rats. Hear Res 1994;73:46–56.
57.
Wu W-J, Sha S-H, Schacht J: Recent advances in understanding aminoglycoside ototoxicity and its prevention. Audiol Neurootol 2002;7:171–174.
58.
Ylikoski J, Pirvola U, Moshnyakov M, Palgi J, Arumäe U, Saarma M: Expression patterns of neurotrophin and their receptor mRNAs in the rat inner ear. Hear Res 1993;65:69–78.
59.
Yoho ER, Thomopoulos GN, Thalmann I, Thalmann R, Schulte BA: Localization of organ of Corti protein II in the adult and developing gerbil cochlea. Hear Res 1997;104:47–56.
60.
Zheng J, Shen W, He DZZ, Long KB, Madison LD, Dallos P: Prestin is the motor protein of cochlear outer hair cells. Nature 2000;40:149–155.
© 2003 S. Karger AG, Basel
2003
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.