IIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIII IIIIIIEGIIIII L'BRARY 3 1293 00791 Mlchlgan State UnMnIty This is to certify that the dissertation entitled OTOTOXICITY IN CHICK EMBRYOS presented by James D. Fikes has been accepted towards fulfillment of the requirements for Ph . D . degree in Pathology 8301' professor Date August 27, 1992 MS U is an Affirmative Action/Equal Opportunity Inuitutt'on 0-12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or bdore date due. II DATE DUE DATE DUE DATE DUE MSU Is An Affirmative ActioNEquel Opportunity lndttution emails-o: OTOTOXICITY IN CHICK EMBRYOS BY James D. Fikes A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1992 ABSTRACT OTOTOXICITY IN CHICK EMBRYOS BY James D. Fikes The most commonly used research animal in testing for ototoxicity is the guinea pig. A chick could possibly serve as an alternative test animal, but it would be more advantageous to use a chick embryo. This research examined the possibility of using the chick embryo as a test animal for ototoxicity. Initially, chick embryos were exposed to a variety of ototoxic aminoglycoside antibiotics and a loop diuretic, ethacrynic acid, to determine if ototoxicity, as characterized by cochlear hair cell loss, was established. Next, the distribution of the aminoglycoside antibiotic, gentamicin, to the avian cochlea was evaluated. Finally, the uptake of gentamicin in relationship to phosphatidylinositol 4,5-bisphosphate (PIPg) by cochlear hair cells was also evaluated. Administration of the aminoglycoside antibiotics alone and in combination with the loop diuretic failed to cause cochlear hair cell loss. Gentamicin was distributed to the avian cochlea early in the treatment period, but intracellular gentamicin was not detected in cochlear hair cells until later in the treatment period. Phosphatidylinositol 4,5-bisphosphate was present in the cochlear hair cells at the earliest time point evaluated. Chick embryos appear to be insensitive to the ototoxic effects of the aminoglycoside antibiotics and ethacrynic acid and the insensitivity does not appear to be due to a lack of presence of PIPE. The insensitivity may be related to insufficient or poorly developed cellular uptake mechanisms in the hair cells, therefore, the chick embryo would not be an appropriate alternative test animal to be used in screening for ototoxins. Dedicated to my parents Clinton H. and Paula M. Carroll. iv ACKNOWLEDGEMENTS I would like to extend a special thanks to Dr. Jim Render, my major professor, for his encouragement and dedication to assisting me in completing this research. I appreciate his insight and direction and the time expended on reviewing my work. I would also like to thank the other members of my committee, Dr. Steve Bursian, Dr. Robert Poppenga, Dr. Willie Reed, and Dr. Stuart Sleight for their suggestions, support and contributions to this research. I am indebted to Carol Ayala, Ralph Common, and Donna Craft of the Department of Pathology, Electon Microscopy Laboratory. Their attention to detail, accommodation of my work into their schedule, and technical advice have been tremendously appreciated. I am grateful to Jeff, Phil, Chris, and Angelo, of the Department of Animal Science, Poultry Science Research and Teaching Center for animal care and assistance in this project. I would also like to thank Drs. Mike Yaeger, Dev Paul, Nina DiPinto, Sue Stejskal, Gail Walter, Leslie Williams, Dave Dorman, John Sullivan, and Bill Valentine for their input and encouragement during my graduate program. TABLE OF CONTENTS LIST OF TABLES ...................................... LIST OF FIGURES ..................................... LIST OF ABBREVIATIONS ............................... INTRODUCTION ........................................ LITERATURE REVIEW ................................... Otic anatomy and embryology ................ Mammalian external and middle ear ..... Avian external and middle ear ......... Embryology of the external and middle ear .......................... Mammalian inner ear .. ................. Avian inner ear ....................... Embryology of the otocyst and cochleovestibular ganglion .......... Cochlear embryology . ................. Vestibular system embryology ......... Synaptogenesis .......... ............. Peripheral auditory electrophysiology ................... Onset of cochlear function ........... Overview of ototoxicology ................. Nonsteroidal anti-inflammatory drugs .............................. Antineoplastic agents ....... .......... Loop diuretics .. ..... . ................ Aminoglycoside antibiotics ................ Chemistry ........................... Pharmacokinetics and metabolism ...... Antibacterial activity ............... Nephrotoxic effects .................. Ototoxic effects ..................... Use of birds in otologic research ......... Aminoglycoside ototoxicity ........... Sound-induced otic trauma ............ Hair cell regeneration ................ vi Page CHAPTER 1: Insensitivity of the chick embryo to the ototoxic effects of aminoglycoside antibiotics and a loop diuretic. Abstract ..... ............................. 67 Introduction .... .......................... 68 Material and Methods ....................... 69 Results ....................... ............ 77 Discussion ................................ 78 CHAPTER 2: Ultrastructural immunocytochemical localization of gentamicin and phosphatidylinositol 4,5-bisphosphate (PIP’) in the cochlea of the chick embryo and the newly atched chick. Abstract ................... ........... .... 90 Introduction .............................. 92 Materials and Methods .... .................. 93 Results .................. ................. 100 Discussion ................................ 114 CONCLUSIONS AND FUTURE STUDIES ...... . ............... 119 APPENDIX ..... .................. . ..................... 125 Introduction ............... ............ .... 125 Materials and Methods ...................... 125 Results ....... .......... ................... 128 Gentamicin studies .................... 128 Kanamycin study ....................... 135 Streptomycin study ...... ........... ... 139 Ethacrynic acid studies ............... 139 Discussion ..... ............. ...... ......... 143 LIST OF REFERENCES .................................. 144 vii Table LIST OF TABLES The year and fungal species or parent antibiotic from which aminoglycoside antibiotics were derived. . ................ Routes of injection and dosages of aminoglycoside antibiotics and ethacrynic acid used to treat chick embryos .......... Mean (1 standard deviation) cochlear hair cell count at 30% of base to apex distance in chick embryos and hatched chicks treated with gentamicin ........................... Routes of administration, dose periods, and dosages of drugs used in dose response studies in chick embryos .................. Mortality in embryonated chicken eggs injected into the air cell with gentamicin at 5.0 to 40 mg/egg/day on incubation days 10 through 17 ........................ Mortality in embryonated chicken eggs injected in the air space with gentamicin at 0.01 to 1.0 mg/egg/day on incubation days 10 through 17 ..... ................... Mortality in embryonated chicken eggs injected in the air space with gentamicin on incubation days 10 through 14 or days 15 through 19 ............................. Mortality in embryonated chicken eggs injected with gentamicin into the allantoic space on incubation days 10 through 17 ................. . .............. Mortality in embryonated chicken eggs injected with gentamicin into the yolk sac on incubation days 10 through 17 ...... viii Page 41 70 110 126 130 131 132 133 134 Table 10. 11. 12. 13. 14. 15. Mean cumulative weight gain (grams) of embryonated chicken 1eggs submerged in a 1.0% gentamicin solution maintained at different temperatures ............ ..... Mortality in embryonated chicken eggs dipped in gentamicin solutions on incubation days 10 through 17 . ............ Mortality in embryonated chicken eggs injected in the air space with kanamycin on incubation days 10 through 17 ..... ..... Mortality in embryonated chicken eggs injected in the air space with streptomycin on incubation days 10 through 17 .......... Mortality in embryonated chicken eggs injected in the air space with ethacrynic acid (EA) on incubation days 10 through 17 ......... .................... Mortality in embryonated chicken eggs injected in the air space with ethacrynic acid and gentamicin on incubation days 10 through 17 . ............ ix Page 136 137 138 140 141 142 Figure LIST OF FIGURES Transverse section of an 19-day chick embryo cochlea at 60% of the base to apex distance .............................. Diagram of proposed mechanism of aminoglycoside-induced ototoxicity ......... Diagram of the basilar papilla from a chick embryo or chick ...................... Photomicrograph of a section of kidney from a hatched chick treated for 9 days with gentamicin at 100 mg/kg with staining of gentamicin using the streptavidin-biotin method with DAB as the chromagen ........... Effect of ototoxic drug treatment on mean (1 standard deviation) hair cell numbers in 18-day chick embryos ....................... Photomicrograph of a section of kidney from a 19-day chick embryo treated with gentamicin by air space injection at 0.1 mg/egg/day on days 10 through 19 of incubation ........... Relationship between integrated optical density (IOD) of basilar papilla sections stained for gentamicin and length of treatment .................................. Photomicrograph of the basilar papilla of chick embryos treated with gentamicin for 5 consecutive days with staining of gentamicin using the streptavidin-biotin method with DAB as the chromogen ....................... .... Photomicrograph of the basilar papilla of hatched chicks treated with gentamicin for 5 consecutive days with staining of gentamicin using the streptavidin-biotin method with DAB as the chromogen ......... . ................. X Page 13 58 72 76 79 80 81 82 83 Figure 10. 11. 12. 13. 14. 15. 16. Page Transmission electron micrograph of a gentamicin immunolabeled section of kidney from a 10-day—old hatched chick after 9 days of treatment with gentamicin at a dose of 100 mg/kg .... ...................... 97 Transmission electron micrograph of a gentamicin immunolabeled section of kidney from a 10-day-old hatched chick after 9 days of treatment with gentamicin at a dose of 100 mg/kg following preincubation of anti-gentamicin serum with gentamicin ... 98 Transmission electron micrograph of a gentamicin immunolabeled section of kidney from a 10-day-old hatched chick, negative control, after 9 days of treatment with deionized water at 0.1 ml/day .............. 99 Transmission electron micrograph of a PIP immunolabeled section of a tall hair cell from a 6-day-old hatched chick, negative control, cochlea after 5 days of treatment with 0.1 ml deionized water/day ........... 101 Transmission electron micrograph of a PIP2 immunolabeled section of tall hair cell stereocilia from a 6-day-old hatched chick, negative control, cochlea after 5 days of treatment with 0.1 m1 deionized water/day following preincubation of anti-PIPZ antibody with an excess of IP3 ............. 102 Transmission electron micrograph of a PIP immunolabeled section of a tall hair cell cuticular plate (CP) from a 6-day-old hatched chick, negative control, cochlea after 5 days of treatment with 0.1 ml deionized water/day following preincubation of anti- IUIQ antibody with an excess of IP3 ........ 103 Transmission electron micrograph of a double immunolabeled section of a short hair cell from a chick embryo on incubation day 17 after 7 days of gentamicin treatment at a dose of 0.1 mg/egg .................... 104 xi Figure Page 17. Transmission electron micrograph of a gentamicin immunolabeled section of a short hair cell from a chick embryo on day 17 of incubation after 7 days of gentamicin treatment at a dose of 0.1 mg/egg ...... ... 105 18. Transmission electron micrograph of a double immunolabeled section of a tall hair cell from a 6-day-old hatched chick after 5 days of gentamicin at 5.0 mg/kg ... 106 19. Transmission electron micrograph of a double immunolabeled section of a short hair cell from a 2-day-old hatched chick after 1 day of gentamicin treatment at 100 mg/kg ..... ............................. 107 20. Transmission electron micrograph of a double immunolabeled section of a short hair cell from an 8-day-old hatched chick after 7 days of gentamicin treatment at a dose of 100 mg/kg .... ..................... 109 21. Photomicrograph of a section of basilar papilla from a 4-day-old hatched chick treated with gentamicin at 100 mg/kg for 3 days .................................... 111 22. Transmission electron micrograph of a section of basilar papilla from a 4-day-old hatched chick treated with gentamicin at 100 mg/kg for 3 days ......... 112 23. Photomicrograph of a section of tegmentum vasculosum from an 8-day-old hatched chick cochlea after gentamicin at 100 mg/kg for 7 days ............. . ....................... 113 xii ABR AC AS BP BSA CAP CP DAB dB DC EA ED EP GFR IOD IP IP IP kHz NSE PIP2 RER RT Sub TBS TTBS TV YS LIST OF ABBREVIATIONS auditory evoked brainstem response air cell allantoic space basilar papilla bovine serum albumin compound action potential cochlear microphonic cuticular plate 3,3 diaminobenzidine tetrahydrochloride decibel direct current ethacrynic acid embryonic day endocochlear potential gentamicin glomerular filtration rate integrated optical density inositol l-monophosphate inositol 1,4-bisphosphate inositol 1,4,5-triphosphate kanamycin kilohertz lysosome neuron-specific enolase phosphatidylinositol 4,5-bisphosphate rough endoplasmic reticulum room temperature streptomycin submersion tris buffered saline tween tris buffered saline tegmentum vasculosum yolk sac xiii INTRODUCTION INTRODUCTION The structure and function of the inner ear, cochlea and vestibular structures, of birds and mammals are comparatively similar (Lewis et al., 1985; Rebillard and Rubel, 1981). The auditory system is relatively mature at birth for human beings and at hatching for chickens, even though development of both structure and function still occurs (Rubel, 1978). The analogy between the avian basilar papilla and mammalian organ of Corti justifies the current routine use of the avian cochlea in studies of general problems of inner ear development and organization (Rubel and Parks, 1988). An understanding of the fundamental development of the inner ear is important in understanding lesions that affect hearing, balance, or posture control. Ototoxicity, although infrequently encountered in clinical practice, may have devastating outcomes (Govaerts et al., 1990). Cochlear effects can range from temporary tinnitus (ringing in ears) to permanent deafness (Brummett, 1980). Vestibular effects include vertigo, nystagmus, and ataxia (Hawkins and Preston, 1975). These clinical symptoms and signs may be temporary (reversible) or permanent (Hawkins and Preston, 1975). Clinically, patients may appear to compensate for permanent vestibular deficits, 1 2 however, there may be little actual improvement of vestibular performance as measured by vestibular function testing methods (Hawkins and Preston, 1975). A long and diverse list of compounds may be considered as ototoxicants. The vast majority of these are pharmaceutical agents such as the aminoglycoside antibiotics, antimalarials, and chemotherapeutic agents (Anniko, 1986; Brummett, 1980; Norris, 1988; Prosen and Stebbens, 1980; Rybak, 1986). Industrial compounds with potential for ototoxicity include solvents such as carbon disulfide (Arlien-Soborg et al., 1981; Miller, 1985; Wood, 1981) and xylene (Aschan et al., 1977; Miller, 1985). Arsenic (Anniko and Sarkady, 1977; Benko et al., 1977; Bencko and Symon, 1977; Miller, 1985), organic mercury (Amin-Zaki et al., 1978; Miller, 1985; Mizukoshi et al., 1975), and lead (Miller, 1985; Ursan and Suciu, 1965) are considered ototoxic metals and metalloids. In addition to ototoxicants, noise should also be considered as an environmental cause of serious harmful effects on hearing (Hamernik et al., 1982). The guinea pig is the test animal of choice in ototoxicologic research because they have morphologic and functional features similar to those of human beings. Both have a dense petrous portion of the temporal bone which precludes the inner ear from being easily accessed for evaluation of otologic lesions. Because of this bone, histologic evaluation requires lengthy decalcification 3 procedures with delicate dissection if the morphology of the inner ear is to be preserved (Micheals, 1988). Alterations in the histologic appearances of some of the tissues occur as a result of the prolonged exposure to solutions used in the decalcification process. Many alterations ascribed to postmortem autolysis are in fact the result of damage by decalcification solutions (Michaels, 1988). To overcome problems associated with the use of the guinea pig for ototoxicologic investigations, investigators have utilized other species of animals or in yitrg methods. Animals utilized include mice (Henry et al., 1981), rats (Osaka et al., 1979), cats (Bernard, 1981), chickens (Lippe et al., 1991), frogs (Kroese and van der Berken, 1982), and parakeets (Hashino et al., 1992). For in yitrg studies, the embryonic inner ear (otocyst) from the mouse (Richardson and Russell, 1991) or chicken (Friedmann, 1965) has been removed at various stages of development and exposed to ototoxic agents in an explant cell culture system. All of these alternatives have advantages and disadvantages. Fertile chicken eggs have been used in alternative ocular irritation tests which have been loosely characterized as an 13 yitrg test. They may provide an opportunity to combine the desirable aspects of using an animal and using an in vitro system. The advantages include the use of a whole live animal which allows for easy comparisons of results and the ability to quickly, cheaply and easily work with the structures of the inner ear. 4 To evaluate the usefulness of chick embryos in otologic research, one needs to have an understanding of the otologic anatomy of mammals and birds, otologic embryology, concepts of ototoxicology, and the use of birds in otologic research. This understanding will provide a basis of knowledge needed to evaluate the distribution of ototoxic drugs to the inner ear and the sensitivity of cochlear hair cells. LITERATURE REVIEW LITERATURE REVIEW OTIC ANATOMY AND EMBRYOLOGY Mammalian External and Middle Ear. The external ear consists of a pinna (auricle) and the external auditory canal (Breazile, 1976; Junqueira et al., 1977). The pinna has a central plate of thin elastic auricular cartilage covered on both sides with tightly attached skin. The external auditory canal is divided into a lateral cartilaginous and medial osseous part. The thin, concave, semitransparent, oval tympanic membrane separates the external and middle ear. Centrally, the membrane is thinner than the periphery. The lateral surface is covered by'a thin layer of epidermis and the medial mucosal surface is covered by simple cuboidal epithelium. Between the epidermal andImucosal layers is a tough connective tissue layer composed of collagenous and elastic fibers. The middle ear consists of the tympanic cavity, which communicates with the pharynx by way of the auditory (pharyngotympanic or Eustachian) tube, and the three auditory ossicles with their associated ligaments and muscles (Junqueira et al., 1977). The auditory ossicles articulate by synovial joints (Junqueira et al., 1977). The, malleus attaches to the medial tympanic membrane surface and then 5 6 articulates with the incus, which in turn articulates with the stapes. The footplate of the stapes attaches to the oval window. The tensor tympani muscle originates in the fossa tensor tympani and inserts on the muscular process of the malleus. Contraction of this muscle tenses the tympanic membrane. The stapedius muscle is the smallest skeletal muscle in the body and originates in the fossa musculae stapedius. It inserts on the muscular process of the stapes. Contraction of the stapedius muscle results in caudolateral movement of the anterior end of the base of the stapes. The main function of the middle ear is to reduce the impedance mismatch between air, the medium of environmental sound wave delivery, and the cochlear fluids (Johnstone, 1988). Energy from sound waves impacting on the tympanic membrane is converted to hydraulic pressure waves in the cochlea by a pumping action of the ossicles at the round window. Contraction of the tensor tympani and stapedius muscles decreases the transfer of sound waves to the inner ear in an attempt to protect the cochlea during prolonged or repetitive loud sounds. Avian External and.Middle Earn The external ear of birds lacks an auricle, but usually possesses specialized feathers, ear (skin) flaps, and.an opercula (Meyer, 1986). The external auditory meatus of birds is a short, curved tube with a circular or oval opening. As in mammals, the tympanic membrane separates the middle and external ears of birds. 7 The middle ear consists of the tympanic cavity, columellar apparatus (avian equivalent to ossicles), and a single columellar muscle (Hodges, 1974). The tympanic cavity is.directly continuous with.the pharynx via the auditory tube. The internal surface of the tympanic membrane is in contact with the columella, which extends across the tympanic cavity and ends at the oval window: The function of the columella is equivalent to the ossicles and the hydraulic transformation quotients of adult bird columellae correspond with those of several mammals (Meyer, 1986) . The columellar muscle, arising from the occipital bone outside the tympanic cavity, passes through a foramen to insert at the apex of the infracolumellar process and the posterior margin of the tympanic membrane. Embryology of the External and Middle Ear. The embryological development of the external and middle ear of mammals and birds is similar. The pinna is formed from the first and second branchial arches (Crary, 1964; Presley, 1984; Wood-Jones and Wen, 1934) . The external auditory meatus begins as an invagination of ectodermal tissue within the first branchial groove. A medial enlargement of the meatus provides the epithelial cover of the tympanic membrane. As other cephalic structures expand, the primary external auditory meatus is forced closed with the epithelial layers left in apposition. Later, secondary extension of the external meatus occurs, and the epithelial layers separate with the lumen once again appearing. Separation of these layers is highly variable among different species of mammals. 8 Species with more mature hearing capabilities at birth may have a fully formed external auditory meatus in late gestation, while in other species (i.e. many rodents), the meatus may not open fully until many days postpartum. The primary external auditory meatus closely correlates with the portion of the meatus surrounded by cartilage in the adult mammal, while the secondary extension relates to that portion surrounded by bone. The middle ear is formed from the first pharyngeal pouch and its associated endodermal tissue (Altmann, 1950). This pouch extends, creating the tubotympanic cavity. This cavity continues to expand by moving in apposition to the external auditory meatus at the point of the future tympanic membrane. The primary extension of the pharyngeal pouch remains constricted and forms the auditory tube (Eustachian tube). The more distal extensions expand to form.the tympanic cavity, tympanic recess, and air cells. With continued differentiation and maturation, specific outpouching of the tympanic cavity occurs along with air cell formation (pneumatization) in adjacent bone. The mucosa of the middle ear consists of different types of epithelial cells including nonciliated, nonsecretory cells; nonciliated, secretory cells (including goblet cells); ciliated cells; intermediate cells: and basal cells. The mucosa of the auditory tube contains ciliated cells and glands and closely resembles that of the respiratory tract. 9 The ossicles of the middle ear are derived from the mesenchymal tissue of the first and second branchial arches (Hanson et al., 1962). The mammalian malleus and incus form from the first arch and the mammalian stapes forms from the second arch. The avian columella, a single ossicle corresponding to the mammalian stapes (Hodges, 1974), is derived from both neural crest and mesoderm (Noden, 1978: Maderson et al., 1982). Ossicles are suspended in mesenchyme until late in development, when the surrounding mesenchyme begins tocdisappear leaving'theIauditory ossicles suspended by ligaments. The tensor tympani, formed from the first pharyngeal arch, is associated with the malleus, and the stapedius, formed from the second arch, is associated with.the stapes. The columellar muscle is considered to be homologous to the stapedius muscle on the basis of its branchial origin (second pharyngeal arch). The columellar muscle and the stapedius muscle are innervated by the facial nerve (Meyer, 1986). The tensor tympani is innervated by the mandibular branch of the trigeminal nerve (Vth cranial nerve). Several types of tissue contribute to the embryologic development of the tympanic membrane. The same tissue which lines the external auditory meatus comprises the outer epithelial layer of the tympanic membrane. This tissue develops into the juxtastromal stratum basal, outer stratum spinosum, stratum granulosum, and stratum corneum. Cephalic mesenchymal tissue trapped between the expanding external auditory meatus and the expanding tympanic cavity forms the 10 fibrous lamina propria. The lamina propria contains capillaries and myelinated and unmyelinated nerves. Ciliated, columnar cells lining the tympanic cavity also cover the medial surface of the tympanic membrane. This particular mucosa is derived from endoderm of the first pharyngeal pouch (Jaskoll and Maderson, 1978). Mammalian Inner Ear; The inner ear is a system of canals and cavities in the petrous part of the temporal bone (Breazile, 1976) and is referred to as the osseous labyrinth. Within the osseous labyrinth is the membranous labyrinth. The osseous labyrinth consists of the vestibule, semicircular canals, and the spiral cochlea. The vestibule lies adjacent to the medial wall of the tympanic cavity. The semicircular canals open into the vestibule on one end and combine to form the crus commune at the other extremity. The canals are uniform in diameter except at one extremity where they dilate to form the ampulla. The osseous cochlea is a coiled structure. The core of the spiral is called the modiolus and the apex is referred to as the helicotrema. An osseous shelf, the spiral lamina, projects into the lumen of the osseous cochlea and spirals around the inner surface of the modiolus. The membranous labyrinth lies within the osseous labyrinth and is separated from it by the perilymphatic space (Breazile, 1976; Junqueira et al., 1977). The utricle and saccule are located within the osseous vestibule. These structures communicate via a narrow, Y-shaped duct whose stalk 11 ends as a blind sac (endolymphatic duct and sac) in the subdural space. The membranous semicircular canals open into the utricle. The membranous labyrinth is lined with simple squamous epithelium, except in regions that differentiate to form specialized receptor organs. ‘These are named the cristae ampullares (within the membranous ampulla) , macula utricularis and macula saccularis (within the utricle and saccule, respectively), and the organ of Corti (within the cochlea). The cristae ampullaris project into the lumen of the membranous ampulla. The epithelium is composed of Type I and Type II hair cells and supporting cells. The epithelium of the cristae ampullaris is covered.by'aIgelatinous cap (cupula) that completely occludes the lumen of the ampulla. The function of the cristae ampullaris is rotational acceleration detection. The macula of the utricle and saccule are rounded areas of sensory epithelium histologically similar to the cristae ampullaris. The surface of the macula is covered with a gelatinous mass containing small calcium carbonate concretions, otoconia. The utricle and saccule are involved in -the detection of gravity, linear acceleration, and vibration. The membranous cochlea is arranged into three chambers, the dorsally located scala vestibuli, the centrally located scala media, and the ventrally located scala tympani. The scala vestibuli and scala media are separated by Reissner's membrane. The scala media and scala tympani are separated by 12 the various cell types of the basilar membrane and the organ of Corti. The organ of Corti (organ of hearing) includes one row of inner hair cells, three rows of outer hair cells, supporting cells, the tunnel of Corti between the inner and outer hair cells, efferent and afferent innervation, and the basilar and tectorial membranes. Endolymph, a fluid similar to intracellular fluid, is found in the scala media, and contains a high concentration of potassium and low concentration of sodium. Perilymph, a fluid found in the scala vestibuli and tympani, has a composition similar to extracellular fluid, including a high concentration of sodium and low concentration of potassium. Avian Inner Bar. In contrast to the coiled mammalian cochlea, the avian cochlea is a relatively short, narrow, slightly curved structure (Hodges, 1974) . When the avian cochlea is sectioned transversely (Figure 1) the basilar papilla (organ of Corti equivalent) is cresent shaped and composed of tall and short hair cells and supporting cells situated on the fibrous basilar membrane (Tanaka and Smith, 1978). Tall and short hair cells have many features which are similar to mammalian inner and outer hair cells, respectively (Whitehead, 1986). The basilar papilla stretches between the superior and inferior fibrocartilaginous plates, separating the scala media from the ventrally located scala tympani. The basilar papilla is covered by the wedge-shaped tectorial membrane» The tegmentum vasculosum forms the lateral wall and roof of the scala media. The tegmentum vasculosum is a 13 TV Figure 1. 'Transverse section of a 19-day chick embryo cochlea at 60% of the base to apex distance. This section contains the basilar papilla (BP), tall hair cells (T), short hair cells (S), tectorial membrane (TM), superior fibrocartilaginous plate (SFP), inferior fibrocartilaginous plates (IFP), scala media (SM), scala tympani (ST), tegmentum vasculosum (TV), and cochlear nerve ganglion (CG). Toluidine Blue. x 140. 14 heavily vascularized structure composed of two highly specialized cell types, light and dark, which are believed to be specialized for active secretion (light cells) and absorption (dark cells). This structure is equivalent to the stria vascularis of mammals (Meyer, 1986). Ganglion cells of the cochlear nerve are located below the superior fibrocartilaginous plate, with myelinated axons extending to the habenula perforata, then distributing as unmyelinated axons across the basilar papilla to hair cells. At the distal (apical) end of the membranous cochlea of birds is another sensory organ, the lagena (Lewis et al., 1985). The lagena consists of a vertically positioned macula covered by a gelatinous otoconial mass. The function of the lagena is unknown, but may be associated with hearing and maintenance of balance (Boord, 1969). Nerve fibers extend from the lagena to the cochlear and vestibular nuclei. As in mammals, three semicircular canals are present in birds with a similar function of detection of angular acceleration (Meyer, 1986). Both ends of each semicircular canal open into the utricle. Each semicircular canal has a dilation (ampulla). Considerable variation exists in the overall configuration and position of the canals in different birds (Meyer, 1986). In the ampulla, a crescent-shaped elevation is present“ .As in mammals, the area perpendicularto the long axis of the canals are covered by sensory epithelium (ampullary crests). Projections parallel to the long axis of the canals are covered by nonsensory, tall 15 columnar cells (plana semilunata). These projections are common in the anterior and posterior semicircular canals of birds. These are infrequent in mammals. The utricle and saccule have sensory epithelium restricted to the macula. Both structures have similar function to their mammalian counterparts. Type I and II hair cells, similar to those found in mammals, are found in the ampullary crests and utricular and saccular maculae of birds. Embryology of the Otocvst and Cochleovestibular Ganglion. The otocyst is the embryologic structure that becomes histologically specialized. as it «differentiates into ‘the cochlea and vestibular system. It begins as a thickened area of surface ectoderm (otic placode) on both sides of the rhombencephalon in early embryogenesis. For example, the otic placode can.be identified in human embryos at approximately 22 days (O'Rahilly, 1963). Adjacent structures, including the rhombencephalon (Harrison, 1936; Jacobson, 1963; Model, 1981; Stone, 1931; Waddington, 1937; Yntema, 1937), nearby mesenchymal cells, and neural crest.cells, appear to influence the formation of the placode. Neural crest cells migrate close to the placode as it begins to thicken. The epithelium of the otic placode continues to thicken, then begins to invaginate to form the otic pit and eventually the otic cup. The rudimentary cochleovestibular ganglion separates from the epithelium as a small cluster of cells that collect at the ventromedial aspect of the otic cup. The period during which separation occurs is short. In the chick 16 embryo, this occurs sometime between the 45th and 71st hours of development (Knowlton, 1967; Meier, 1978), but.not after 72 hours (Whitehead and Morest, 1985a). Although separated from the otic placode, cells of this embryonic ganglion are mitotically active and continue to divide to form neurons until embryonic day 6 in the chick embryo (D'Amico-Martel and Noden, 1983). All of the neurons of the cochlea, and the vast majority of the vestibular neurons are derived from otic placode (D'Amico-Martel and Noden, 1983; Yntema, 1937). Some neurons in the vestibular ganglion are derived from the neural crest. Schwann cells and satellite cells in the ganglion are entirely of neural crest origin. As the rim of the otic cup fuses, the otocyst (otic vesicle) is formed (Whitehead, 1986). This structure appears as a hollow, almost spherical, ball that forms the cochlear and vestibular membranous labyrinths. The epithelium of the otocyst, through a complex progression of cytodifferentiation, forms the numerous cell types that make up the membranous labyrinth (Whitehead, 1986). Initially, the otocyst is without innervation, but synapses occur as nerve fibers grow from the ganglion cells and actively enter the epithelium. Because of differential growth, the spherical otocyst begins to contract in some areas and distend in others. An early appearing constriction delineates the dorsal utricular bulge from the ventral saccular region. The cochlea develops from the saccular region as a ventral evagination of thick pseudostratified columnar epithelial cells. The utricular l7 bulge envaginates a small area of thin cuboidal epithelium that forms the endolymphatic duct. Next, the utricular epithelium is pulled into dorsally located folds which cavitate as the semicircular canals. Cochlear Embryology. The cochlear evagination formed from the saccular region elongates ventrally and medially to form the lagena in lower vertebrates, or as a straight tube housing the basilar'papilla.in.birds~ Its distal end contains the macula lagena. In mammals, the ventrally extending cochlear evagination becomes relatively long and begins to form coils. The connection with the saccule continues as the narrow ductus reuniens. There appears to be considerable overlap in the times of generation of different cell types within the cochlea. However, there. is a spatial gradient of hair cell and supporting cell cytodifferentiation that occurs along the length of the cochlea (Ruben, 1967). Cells at the apex undergo terminal mitosis before more basilar regions. Once the membranous labyrinth attains all of its principal components, the surrounding mesenchyme condenses to form the bony labyrinth (Knowlton, 1967). Mature hair cells are not attached to the basilar lamina like supporting cells. As hair cells differentiate, they lose their basal cytoplasmic attachments (Orr, 1975), as well as their attachments to adjacent cells (Ginzberg and Gilula, 1979). Primitive hair cells in the cochlear epithelium lose their basal attachments and extend toward the luminal surface 18 as they withdraw their basal extensionsu During later stages, nerve fibers enter the epithelium and attach to the withdrawing basal processes of these cells as they migrate to the surface. It is at this time hair cells become apparent by electron microscopy (Whitehead and Morest, 1985a; Whitehead and Morest, 1985b). These neuroepithelial junctions have the morphology of immature synapses, and synaptic bodies appear for the first time in the newly differentiated hair cells (D'Amico-Martel and Noden, 1983). Synaptic bodies, also called synaptic bars, are presynaptic differentiations in the form of invaginations (Ades and Engstrom, 1974). Hair cells do not seem to require innervation for their differentiation and survival (Ard et al., 1985; Fell, 1938: Van de Water, 1976). However, hair cells may provide some trophic influence upon vestibulocochlear ganglion cells because the survival of ganglionic neurons in culture is dependent upon the presence of peripheral target tissue (Ard et al., 1985). Normal differentiation of the vestibular and cochlear hair cells and cartilaginous otic capsule do depend upon reciprocal interactions between the growing otocyst and surrounding mesenchyme (Van De Water, 1983). Hair cells are differentiated from undifferentiated supporting cells when they are confined.to the luminal surface and form stereocilia and kinocilia from precursors of the microvilli. The ciliary bundle of hair cells in the mouse matures when the kinocilia are lost (Kikuchi and Hilding, 1965). The tunnel separating inner and outer hair cells in 19 mammals forms as pillar cells differentiate from the rather tall supporting cells located between the immature inner and outer hair cells (Kikuchi and Hilding, 1965; Larsell et al., 1944; Lenoir et al., 1980). The tunnel forms as a slit of extracellular space between the tall supporting cells and expands as a triangular shaped structure that widens to form the tunnel of Corti. As the tunnel forms, the expanding bases of the pillar cells cause the course of some afferent and efferent fibers leading to outer hair cells to be redirected as they cross the tunnel (Ginzberg and Morest, 1984). With continued maturation, the bases of the tall supporting cells bordering the tunnel widen and the free sides become convex. The cells now become recognizable as pillar cells. At the same time, other cytodifferential changes in the organ of Corti, including the differentiation of the phalangeal cells, the appearance of the space of Nuel, the opening of the internal spiral sulcus, a thinning of the basilar membrane (Anniko, 1980), and an increase in the thickness and density of the tectorial membrane (Lenoir et al., 1980; Pujol and Marty, 1970) are occurring. The basilar papilla (Fermin and Cohen, 1984) and organ of Corti (Hilding, 1968; Larsell et al., 1944; Li and Ruben, 1979; Nakai and Hilding, 1968; Pujol and Marty, 1970; Sher, 1971) do not differentiate synchronously throughout their lengths. In general, hair cells and supporting structures differentiate earlier in basal regions and later in the apical region. An exception to this general statement is the 20 relatively late differentiation that occurs in the extreme basal area in some species (Larsell et al., 1944). It is unclear if a similar pattern of differentiation occurs in regards to innervation. Intraepithelial cochlear nerve fibers have been clearly identified in basal regions containing' differentiated hair' cells, but not in apical regions with undifferentiated hair cells (Larsell et al., 1944; Sobkowicz et al., 1975). Nerve fiber growth into the sensory epithelium may«occur first in mid-basal regions (Sher, 1971). An examination of other morphologic features may reveal a slightly different pattern of differentiation. Results of an examination of the generation times of ganglion cells and hair cells from different parts of the cochlea indicates that the developmental patterns of ganglion cells and hair cells are different rather than similar (Ruben, 1967). The first hair and supporting cells undergoing terminal mitosis occupy the apical region while the first ganglion cells formed are located in the basal region. Subsequently generated neurons are located in apical locations. The pattern of ganglion cell generation match the pattern of hair cell differentiation, but not the pattern of hair cell generation. Presumably, the time between terminal mitosis and cytodifferentiation is longer for apical hair cells than basal cells. Vestibular System Embryology. The vestibular system originates within the otocyst (Anniko, 1983). The developmental sequence of the vestibular system in birds is 21 very similar to that observed in man (Anson, 1934; Anniko, 1983). Morphogenesis of the vestibular system occurs in the chidk embryo during embryonic days (ED) 3.5-6.5 (Knowlton, 1967). The endolymphatic appendage develops from an outgrowth of the dorsal aspect (future utricular portion) of the otocyst. This structure develops into the endolymphatic duct and sac. The endolymphatic appendage is delineated from the future vestibular tissue of the otocyst by a downward directed edge, the future position of the utriculoendolymphatic valve (utriculosaccular chamber). The endolymphatic appendage elongates with general expansion of the otocyst as a whole. The endolymphatic appendage divides into the proximally located ductus and distally located expansion of the saccus (Bast and Anson, 1945). The endolymphatic sac continues to extend dorsally into the differentiating meningeal tissue. With continued fetal development, the dura containing the endolymphatic sac migrates downward forcing the distal end of the thinned part (isthmus) of the endolymphatic duct and the proximal part of the sac to change from a straight to curved configuration. 'Fhis leaves the apex of the endolymphatic sac, which was dorsal in the fetus, directed dorsocaudally in the adult. The semicircular canals appear as flattened, concave outpocketings of the utricular part of the otic vesicle. The formation order for the semicircular canals is anterior, posterior, and lateral (Bast et al., 1947). The central 22 aspects of the walls of these outpocketings eventually become apposed to each other at the baseu The apposed embryonic wall tissue is reabsorbed creating the semicircular canal. One end of each canal dilates to form the ampulla. The first anlage of all the later sensory end organs is a thickening of the medial wall of the otocyst near the statoacoustic ganglion called the macula communis (Anniko, 1983). Differentiation of the neuroepithelium of cristae and maculae start at about the same time (Altmann, 1950). In the chick embryo, neural connections between the statoacoustic ganglion and future hair cells have been identified after 78 hours of incubation (Proctor and Lawrence, 1959). The anterior cristae and macula sacculi become separate units first (ED 5.5-6) followed by the posterior cristae and macula neglecta (ED 6-6.5) and, finally, the lateral cristae, macula utricle and lagena, and the papilla basilaris (ED 6.5-7). Hair cells and supporting cells are separated earlier in the posterior and anterior cristae (ED 5-5.5) than in the lateral cristae, macula utriculi, neglecta, and lagena. Histologic differentiation of hair cells is characterized by argentophilic transformation of cytoplasm (ED 5.5-8) , rounding of the nuclei, and the cells become pear shaped. stereocilia are identified by ED 8 and become longer through ED 10. The cristae and macula have a rather mature appearance grossly by ED 10. 23 Synaptogenesis. Synaptogenesis of the cochlea involves both afferent and efferent innervation. Synapses between hair cells and the peripheral processes of the cochlear ganglion are immature when first formed. Both the nerve fiber and the hair cell undergo a series of structural modifications with the end result being a synapse. During early synaptogenesis in cats and rats, afferent nerves posses many terminal branches in areas below the hair cells or beyond the base of the hair cell toward the cochlear duct (Perkins and Morest, 1975; Whitehead and Morest, 1985a). In chick embryos, most of these extraneous branches disappear as the remaining nerve fibers develop large, preterminal swellings below the hair cells. During this period, hair cells exhibit an increased number of synaptic bodies.in.embryonic chicks (Hirokawa, 1978) and. mice (Sobkowicz et al., 1982). This results in a distribution of synaptic bodies to sites not adjacent to the nerve endings. During final afferent synaptogenesis, the heterotopic distribution of synaptic bodies is replaced by a pattern in which all synaptic bodies are situated presynaptically adjacent to a synapse (Whitehead and Morest, 1985b). This change in distribution of synaptic bodies in the final phase of synaptogenesis is apparently due to a decrease of synaptic bodies not adjacent to the synapse (Favre and Sans, 1979; Sobkowicz et al., 1982). Nerve fibers in late synaptogenesis lose their presynaptic swellings in birds (Whitehead and Morest, 1981) and mammals (Ginzberg and Morest, 1983; Lorente, 1937; Perkins 24 and Morest, 1975) and appear as gnarled endfeet. Some afferent terminals committed to synapses may degenerate or decrease the area of their synaptic contact (Pujol et al., 1980; Whitehead and Morest, 1985b) with some endings even competing for postsynaptic sites (Favre and Sans, 1979). In birds and mammals, the hair cells have efferent innervation from crossed and uncrossed olivocochlear nerve fibers (Ginzberg and Morest, 1983). The establishment of efferent innervation may be the final neuronal development as the cochlea nears final maturation and occurs for the most part, after afferent innervation is well underway. In fact, the formation of efferent synaptic endings coincides with the period when cochlear function begins in the chick embryo (Rebillard and Rubel, 1981; Whitehead and Morest, 1985b). In chick embryos, the efferent fibers extend from the medulla into the developing cochlea 3 in: 5 days after the afferent fibers (Cohen and Fermin, 1978; Whitehead.and.Morest, 1985b) . These efferent projections apparently follow existing afferent fibers to the hair cell regions (Whitehead and Morest, 1985a; Whitehead and Morest, 1985b). In the basilar papilla of chick embryos, small efferent synapses are formed on tall hair cells and their subjacent afferent dendrites, while large efferent endings contact short hair cells. Efferent synaptic endings form in chick embryos during the intermediate stages of afferent synaptogenesis. During this period, afferent fibers are remodeled into endings with.mature sizes and shapes. 25 Efferent synaptogenesis in mammals occurs predominately at.a postnatal time, after the inner and outer hair cells have differentiated (Ginzberg and Morest, 1983; Ginzberg and Morest, 1984). During postnatal development, efferent synapses with inner hair cells decrease and form synapses only on afferent dendrites (Pujol et al., 1980). Synchronously with the formation of efferent synaptogenesis, outer hair cells lose all synaptic bodies formed during early afferent synaptogenesis and develop subsynaptic cysternae opposite the efferent nerve terminal (Pujol et al., 1979). Peripheral Auditory Electrophysiology. The variety of electrophysiologic methods used to assess cochlear function used produce a diverse assortment of research results to understand. The resting endocochlear potential (EP) consists of two components: a positive portion thought to be produced by an electrogenic sodium-potassium pump, and a negative component produced by potassium diffusion or a leakage of current from cells of the organ of Corti (Moller, 1983). The cochlear microphonic (CM) most likely originates from the hair cells, and recordings from the round window reflect the activity of mainly the outer hair cells in the base of the cochlea (Moller, 1983). The compound action potential (CAP) is a sound dependent potential reflecting excitation and synchronization of populations of auditory nerve fibers (Dallos, 1981). The response is most.prominent following transient stimuli such as clicks and the potential is composed of two peaks, named N1 26 and N2. The magnitude of the N1 response is a function of both the stimulus intensity and the number of fibers firing synchronously. The N} amplitude and latency are thought to reflect the integrity of the cochlea and eighth nerve, whereas the N2 component may originate from more central structures. The auditory evoked brain response (ABR) consists of a series of waves during the first 10 msec following a click or pure tone stimulus (Jewett, 1970). The ABR is extracted from the background electroencephalogram activity by computer and represents sequential activation of the auditory neuroaxis in the brain stem. The N1anuitg components of the CAP are the first two waves of the ABR. The tuning curve is a plot of neurons firing above threshold at various frequencies (Dallos, 1981; Moller, 1983) . The neural response at various frequencies is not the same. A tuning curve consists of a low-frequency tail portion that indicates hearing response only to high-intensity sounds and an extremely sharp tuned "tip" region. The latter is characterized by an extremely sudden transition from the high- threshold tail region to a narrow region where the nerves or receptor cells increase their firing rate abruptly at tones of low intensity near the characteristic frequency. At frequencies above the characteristic frequency, there is again an abrupt and rapid rise in the threshold of the neuron. Onset of Cochlear Function. It is likely that no single change is responsible for the onset of hearing in birds due to the multitude of structural, biochemical, and physiologic 27 changes occurring simultaneously (Rubel and Parks, 1988). These events are well synchronized despite their diversity with the product being a functional inner ear. The avian basilar papilla is responsive to sound and develop auditory capabilities during the last half of embryogenesis. The earliest electrical potentials recorded from the basilar papilla of the chick embryo in response to sound stimulation were first seen at ED 10, but could only be elicited by a very intense stimuli (Saunders et al., 1973). This developmental stage is 2 to 3 days after the first definitive afferent nerve-hair cell synapses are formed (Cohen and Fermin, 1978; Hirokawa, 1978; Whitehead and Morest, 1985b). By ED 11-12, a neural component appears (Saunders et al., 1973). Averaged evoked responses can be recorded from the brain stem. Intensities over 100 dB are still required, however, and only relatively low-frequency, long-duration tone bursts are effective. This event coincides with at least one measure of biochemical differentiation. Whitehead et a1. (1982) described hair cells of the basilar papilla beginning to demonstrate neuron—specific enolase (NSE) on ED 10. In chick embryos, central responses evoked by tone bursts can be recorded at the level of the first central synapse, the nucleus magnocellularis, as early as ED 11 (Vanzulli and Garcia-Austt, 1963). The nucleus magnocellularis is considered homologous to the mammalian anterioventral cochlear nucleus (Boord, 1969). The response was poorly organized and slow until ED 15 when the central responses became more 28 synchronized with a shorter duration in conjunction with the onset of click-evoked activity (Saunders et al., 1973). Also during this period, behavioral responses to sound.can first be elicited (Jackson and Rubel, 1978). The amount and distribution of NSE increases dramatically during this time frame (Whitehead et al., 1982). Prior to ED 16-17, the auditory system is mainly capable of responding to loud, low- frequency soundsw This is followed by'a decrease in threshold intensity (increased.sensitivity) across.all frequency ranges, primarily due to middle ear clearing (Rubel and Parks, 1988). Morphologically, onset of click-evoked activity and surge in NSE coincides with a transition from mid to late synaptogenesis. During this time, the basilar papilla exhibits considerable remodeling of the afferent synaptic endings and the beginning of efferent synaptogenesis (Whitehead and Morest, 1985b). Sharpening of the tuning curve observed during prenatal development of the chick embryo correlates with establishment of efferent innervation of the short hair cells and the final stages of maturation of afferent synapses (Rebillard and Rubel, 1981). As mentioned previously, the differentiation of hair cells and the development of at least some neural features in.both.birds and mammals generally' proceeds from. basal to apical regions (Fermin and Cohen, 1984; Li and.Rubin, 1979; Sobkowicz et al., 1975). It has been well established that once mature, the basal cochlea detects high frequencies while the more apical regions detect lower frequencies. However, the earliest 29 frequency responses generated in both birds and mammals have been low-frequency stimuli while high-frequency responses mature relatively late (Brugge et al., 1978; Konishi, 1973; Moore and Irvine, 1979; Rebillard and Rubel, 1981; Saunders et al., 1973). This creates a paradox between the structural maturation sequence of the organ of Corti and basilar papilla and the development of frequency selectivity. The frequency limitation probably is not the result of immaturity of sound conduction through the immature outer and middle ear because it holds true even when auditory stimuli are applied after clearing of the external and.middle ears (Rebillard and Rubel, 1981). Evidence suggests that the frequency organization of theIcochlea shifts.during development (Rubel and Ryals, 1983). Chick embryos exposed to high intensity pure tones that cause discrete foci of hair cell damage have regions of hair cell loss that differ along the basilar papilla from basal to apical regions for each of the tones applied. From this it appeared that basal regions mediate lower frequency hearing during earlier stages of hearing development than during later stages. OVERVIEW OF OTOTOXICOLOGY Prosen and Stebbins (1980) defined ototoxins and vestibulotoxins as agents that affect hearing and balance, respectively. Many toxic compounds, however, affect both systems and may be referred to as ototoxicants. Ototoxicants are agents which, when administered, lead to hearing 30 impairment and corresponding structural damage to the peripheral auditory system or precipitate an impaired sense of balance and structural damage to the peripheral vestibular system. Ototoxic effects on hearing can be classified as conductive or sensorineural hearing deficits. Conductive hearing deficits are the result of malformation of the outer and/or middle ear sound wave conductive pathways to the cochlea. Thalidomide, quinine, vitamin A, and retinoic acid are a few of 'the examples of this type of ototoxicant (Granstrom, 1990; Schuknecht, 1974). Sensorineural hearing deficits are the most common type of ototoxic effect and are due to a loss of the sensory epithelium (hair cells) or damage to central nervous system auditory components. By the 18805, it was known that quinine, salicylates, and oil of chenopodium produced both temporary and permanent hearing impairment and vestibular disturbances (Hawkins, 1976). With the reports of the auditory and vestibular effects of the first aminoglycoside, streptomycin, in 1945, ototoxicity began to receive significant attention (Hinshaw and Feldman, 1945). Reports of both transient and permanent hearing loss following the use of the loop diuretic, ethacrynic acid, began to appear in 1965, shortly after it was introduced into clinical practice (Bosher, 1980a). Since the loop diuretics have a unique ototoxic mechanism of action separate from the aminoglycosides, this reemphasized the susceptibility of the inner ear to ototoxicants. 31 Since 1988, the drugs most commonly associated with ototoxicity are the aminoglycoside antibiotics, nonsteroidal anti-inflammatory agents, loop diuretics, and antineoplastic agents (Norris, 1988). Their structures are quite diverse, as are their ototoxicologic signs, primary otic target tissue, and the type of change they induce (Schacht and Canlon, 1988). Nonsteroidal anti-inflammatory drugs and antineoplastic drugs will be briefly reviewed because of their current clinical relevance and classic association with ototoxicity. Nonsteroidal Anti-Inflammatory Drugs. This group of drugs includes aspirin, sodium salicylate, and methyl salicylate, as well as newer agents such as ibuprofen and naproxen. The nonsteroidal anti-inflammatory drugs such as naproxen and ibuprofen have a lower incidence of ototoxicity, but when it occurs, it is frequently irreversible (Miller, 1985; Chapman, 1982). The most common ototoxic symptoms are reversible tinnitus (a high-pitched ringing in the ears) and hearing deficits (Douek, et al., 1883: Miller, 1985). Little progress has been made on the mechanism of action of salicylates. Early research revealed mitochondrial vacuolation in cells of the stria vascularis and outer hair cells (Covell, 1936). Falbe-Hansen (1941) proposed that salicylates increased perilymph production, inducing a cochlear hydrops. However, histologic and/or ultrastructural changes have been rarely reported (Bernstein and Weiss, 1967; Deer and Hunter-Duvar, 1982; DeMoura and Hayden, 1968; Falk, 1974; Morrison and Blakley, 1978). Flattening and destruction 32 of the epithelial elements of the organ of Corti in the guinea pig have been reported (Aly et al., 1975). Several biochemical mechanisms of action have been proposed for this group of drugs. The anti-inflammatory properties of this group of drugs results from the inhibition of cyclooxygenase, an enzyme catalyzing the first step in the synthesis of prostaglandins from arachidonic acid. Aspirin lowers the rate of prostaglandin synthesis in the cochlea as indirectly indicated by the lower levels of prostaglandins in perilymph (Escoubet et al., 1985). A causal relationship between cyclooxygenase inhibition and ototoxicity has been proposed (Brown and Feldman, 1978; Rybak, 1986), but evidence for this is unsubstantiated. Salicylates may alter membrane potentials and cellular energy production resulting in altered propagation of cochlear and auditory nerve action potentials. Salicylates inhibit cochlear transaminase and dehydrogenase systems (Silverstein et al., 1967) and acetylcholinesterase activity in efferent nerve endings (Ishii et al., 1967). While ATP and phosphocreatine content of the stria vascularis and cochlear nerve increase, reductions in ATP concentrations of Reissner's membrane are reported after salicylate treatment (Krzanowski and Matschinsky, 1971). In addition, vasoconstriction has been found experimentally in capillaries of the suprastrial spiral ligament, the stria vascularis, the tympanic lip and basilar membrane (Hawkins, 1973). 33 Antineoplastic Agents. Ototoxic antineoplastic agents include cisplatin (De Conti et al. , 1973) , 6-aminonicotinamide (Herter et al. , 1961) , nitrogen mustard (Cummings, 1968) , vincristine (Mahajan et al., 1981), vinblastine (Serafy et al., 1982), and misonidazole (Abratt and Blackburn, 1980). cisplatin induces a histologic change consisting of outer hair cell loss in the basal turn of the cochlea (Nakai et al., 1982, Komune et al. , 1981; Konishi et al. , 1983) that is strikingly similar to that seen with aminoglycoside antibiotics. ‘Ultrastructural changes include supporting cell damage and irregular stereocilia of inner and outer hair cells (Estrem et al. , 1981) . In addition, vestibular toxicity (Kobayashi et al., 1987; Tange and. Vuzevski, 1984) and degeneration of marginal, intermediate, and basal cells of the stria vascularis have also been reported (Kohn et al., 1988; Tange and‘Vizevski, 1984). Cochlear changes in human patients with. cisplatin-induced. hearing loss include large, fused stereocilia and damage to the cuticular plate of the basal outer hair cells, with degeneration of spiral ganglion cells and cochlear neurons (Strauss et al., 1983; Wright and Schaefer, 1982) . A proposed mechanism of action involves the inhibition of adenylate cyclase (Kopelman et al., 1988). Some ototoxic antineoplastic compounds are rarely used today. 6-Aminonicotinamide produces a number of toxic side effects such as irreversible vertigo, hearing deficits, and tinnitus (Herter et al., 1961). The histologic changes in human beings and cats induced by nitrogen mustard are 34 characterized by severe loss of hair cells in the basal and middle turns of the cochlea with the apex being spared (Schucknecht, 1964, Cummings, 1968). vincristine produces degenerative effects in the spiral ganglion and organ of Corti hair cells (Serafy and Hashash, 1981) while vinblastine produces degenerative changes only in hair cells (Serafy et al., 1982). Apparently replacing the formyl group on vincristine with the methyl group on vinblastine has a sparing effect on the spiral ganglion” Only audiologic changes have been reported with misonidazole (Abratt and Blackburn, 1980). Loop Diuretics“ The loop diuretics are a widely used and important component of the therapeutic approach to a variety of disease processes. Loop diuretics include furosemide, ethacrynic acid, piretanide, bumetanide, azosemide, ozolinone, and indacrinone (Jacobson and Kokko, 1976). Although chemically dissimilar, members of the group have a common mode of action in producing diuresis, but, the specific ototoxic mechanism of action of the loop diuretics is still unknown. The development of acute, reversible deafness following the intravenous use of ethacrynic acid in man was first described by Maher and Schreiner (1965). Each of the active loop diuretics has been shown to have an adverse effect on cochlear function in experimental animals and most have been shown to be ototoxic in humans (Rybak, 1986). One unique feature of furosemide is the lack of an ototoxic effect on pigeons suggesting a possible fundamental difference in inner 35 ears between birds and mammals (Schermuly et al., 1983). In human beings, the clinical effects of loop diuretic- induced ototoxicity are those of hearing loss and vestibular disturbances (Michaels, 1988). These effects are usually reversible, but can become permanent (Klinke et al., 1981). High doses or repetitive administration of ethacrynic acid have been shown to produce permanent hearing loss and damage to the hair cells in the organ of Corti (Mathog et al., 1970) which may result from a direct action on the hair cells or be secondary' to prolonged. changes in the stria vascularis. Furosemide-induced hearing loss occurs most frequently in patients with concomitant renal disease and is associated primarily with intravenous administration at rates exceeding 4 mg/min (Gallagher and Jones, 1979). The hearing loss associated with furosemide can have a latency of onset up to 6 months with progressively higher thresholds of hearing (Quick and Hoppe, 1975). Ethacrynic acid ototoxicity has a definite latent period after administration (Brown, 1975; Brown, 1981; Brown and McElwee, 1972). A possible explanation for the delay is that metabolism to a more toxic intermediate is necessary. Experimentally, ethacrynic acid and furosemide produce a dose-related reduction in endocochlear potential and/or alteration in endolymphatic ion concentration with the concomitant reduction in cochlear microphonic and subsequent by depression of auditory-evoked responses (Brown, 1975; Brown, 1981; Brown and McElwee, 1972; Brusilow, 1976; Kusakari 36 et al., 1976; Rybak and Morizono, 1982; Silverstein and Yules, 1971; Spoendlin, 1988). Loop diuretics appear to block the potassium chloride cotransport system that moves potassium and chloride out of the stria vascularis into the endolymph (Marcus et al., 1983; Santi and Lakhani, 1983). This may explain the endolymph electrolyte abnormalities such as reduced concentration of potassium (Bosher, 1979; Rybak and Morizono, 1982) and chloride (Brusilow, 1976) and an elevation in sodium concentration (Brusilow, 1976) induced by the loop diuretics. Although the mechanism of action of loop diuretics in producing' ototoxicity' is 'unknown, the jproposed. molecular effects fall into the following three general categories (Bosher, 1980a,b): (1) Energy utilization. The stria vascularis is responsible for the positive direct current (DC) endocochlear potential and maintenance of the endolymph electrolyte balance. The generation of the endocochlear potential has been shown to be dependent upon a sensitive Na"/K+ membrane ATPase pump which led to the suggestion that loop diuretics may produce their ototoxic effect by inhibition of strial NaUQC ATPase activity (Prazma et al., 1972; Thalmann et al., 1973). This hypothesis is consistent with the alterations in strial ATPase and phosphocreatine dynamics observed during ethacrynic acid ototoxicity. However, 1 vivo studies do not support this hypothesis (Kusakari et al., 1976, 1977). The hypothesis that loop diuretic induce ototoxicity via 37 inhibition of strial adenylate cyclase is not generally accepted (Kusakari et al., 1976; Marks and Schacht, 1982; Paloheimo and Thalmann, 1977). (2) Decrease in membrane permeability: Changes noted in the stria vascularis during ethacrynic acid ototoxicity suggest alterations in the permeability of the endolymphatic membranes. Direct evidence of changes in membrane permeability are lacking (Bosher, 1980a,b). However, Bosher (1980b) and Forge (1981) reported disruption and alterations in the limiting membrane of the surface coat of marginal cells, possibly reflecting a functional change of the endolymphatic surface membrane of strial marginal cells. (3) Depression of oxidative metabolism (energy generation). As previously mentioned, stria vascularis metabolism is believed responsible for the endocochlear potential whereas the metabolic activity of the organ of Corti is thought to represent the cochlear microphonic potential. Ethacrynic acid has been shown to reduce the cochlear microphonic potential in two phases of decline (Prazma et al., 1972). The two phases were attributed to an initial change in potassium ion concentration inside the cochlear duct that altered. the cochlear microphonic potential followed by a decrease in glycolysis in hair cells. Because of low glycogen storage in the hair cell, altered microphonic potentials might be expected to persist for some time, even after ion transport within the cochlear duct has been reestablished. The effect of ethacrynic acid on the 38 organ of Corti could thus be independent of any effect on the stria vascularis (Miller, 1985) and this is supported by Horn et al., (1978) who suggested that the acute depression of the cochlear microphonic potential was due to a decrease in energy production within the organ of Corti. A reversible impairment in the electron transfer system of the hair cell similar to that seen in renal mitochondria (Cunarro and Weiner, 1978) has been proposed as the mechanism of furosemide ototoxicity (Tamura, 1978). Evidence exists that ethacrynic acid is ototoxic in the parent form (Fox and Brummett, 1974) and as the cysteine metabolite (Brown, 1975). Koechel (1981) demonstrated a direct correlation between the release of ethacrynic acid from the cysteine conjugate _ig yiLrg and the ototoxic potential in guinea pigs. Koechel (1981) also demonstrated a direct correlation between a series of ethacrynic acid thiol adducts and their ototoxic potential. In addition, the cysteine conjugate of ethacrynic acid is 3.1 to 3.3 times more potent at suppressing the N1 potential than ethacrynic acid alone (Brown, 1975). This may be explained by ethacrynic acid having a greater protein binding capacity than the cysteine conjugate, allowing more of the cysteine conjugate to diffuse across the blood/cochlear barrier (Fox and Brummett, 1974). Furosemide concentrations within the inner ear can be reduced and the ototoxicity ameliorated or prevented by administration of probenecid, an inhibitor of organic acid transport (Rybak et al., 1984). This suggests that the 39 diuretic uptake mechanism in the kidney, consisting of organic acid active transport from the blood to its site of action, may also be the same uptake mechanism in the cochlea. Loop diuretic-induced morphologic changes are reported in both the cochlear and vestibular systems. Cochlear changes occur primarily in the stria vascularis of the basal cochlea (Santi and Lakhani, 1983; Arnold et al., 1981). This change is characterized by edema between and within cells of the stria vascularis. With excessive doses of loop diuretics, there is also damage to the outer hair cells of the basal cochlea (Matz, 1976). ‘Vestibular changes are characterized.by cyst formation in the sensory epithelium of the posterior semicircular canal and the saccular macula (Matz, 1976). The earliest vestibular changes are visible only by electron microscopy, with later damage visible by light microscopy (Forge, 1980). The damage to the sensory epithelium correlates well with alterations in vestibular electrophysiology (Brummett et al., 1977). Although numerous methods have been utilized in the investigation of loop diuretic-induced ototoxicity, the exact mechanism is still unknown. Of the proposed biochemical effects, alterations in energy production by the hair cells and energy utilization in the stria vascularis may account for the separate effects on the vestibular and cochlear systems, respectively, following administration of ethacrynic acid while alterations in the electron transfer system within hair cells may be responsible for furosemide ototoxicity. AMINOGLYCOSIDE ANTIBIOTICS The aminoglycoside antibiotics began to appear in 1957 with the introduction of kanamycin (Pancoast, 1988). This group of antibiotics provided the beginning of an effective method for treating gram negative infections. With the advent of newer agents in this group, such.as amikacin, with activity against Pseudomonas aeruqinos_a, the aminoglycosides became the standard for the treatment of gram negative infections (Kawaguchi, 1976). Aminoglycoside antibiotics are a widely used group of antibiotics, with about three million doses being given per year in the United States (Pancoast, 1988). This usage has grown despite the availability of expanded spectrum beta- lactam antibiotics with equal activities (Davies, 1986) . This wide use has also brought out the problems of toxicosis, super infections, and bacterial resistance. Interest.in the toxic sideteffectslhas.generated interest ixithe development.of other antibiotics with the same spectrum of activity, but with less associated toxicity: Many of these antibiotics are now available (Levine et al., 1985; Moellering, 1986; Neu, 1983; Neu, 1986; Nichols, 1986). Chemistry. Streptomycin, the first aminoglycoside discovered, was isolated from the fungus Streptomvces grisens (Table 1). Other members of the aminoglycosides were also discovered by soil searching techniques. In 1963, the gentamicin complex (gentamicin C“ Cay,(§) was isolated from Micromonospora purpura (Siegenthaler et al., 1986). The 40 41 Table 1. The Year and Fungal Species or Parent Antibiotic FromflWhich Aminoglycoside.Antibiotics Were Derived (Siegenthaler et al., 1986). Fungal Species or Year Antibiotic Parent Antibiotic 1944 Streptomycin Streptomyces grisens 1949 Neomycin Streptomyces fradiae 1957 Kanamycin Streptomvces kanamyceticus 1963 Gentamicin Micromonospora purpurea 1967 Tobramycin Streptomvces tenebrarius 1970 Sisomicin Micromonospora inyoensis 1972 Amikacin Kanamycin A 1975 Netilmicin Sisomicin 42 suffix "mycin" designates an antibiotic isolated from Streptomyces sp. while "micin" indicates an isolate from a Micromonospora sp. Later aminoglycosides were chemically synthesized. These include amikacin (the first synthetic), paromomycin, tobramycin, Sisomicin, and netilmicin (Kawaguchi, 1976; Miller et al., 1976). The core aminoglycoside molecule consists of two or more cyclic amino sugars bound by a glycoside linkage to a central hexose. All aminoglycosides are aminocyclitols, but not all aminocyclitols are close enough analogs to the aminoglycosides to possess a similar antibacterial activity. An example of this is the antibiotic spectinomycin. The aminoglycosides are highly water soluble polar cationic compounds with optimal activity at a pH of 6 to 8. Their antibacterial activity is strongly curtailed by acid pH and divalent cations. The agents do not perform well in the acid environment of bronchial secretions or abscesses, or in the presence of necrotic tissue and large quantities of organic material containing divalent cations (Bodem et al., 1983) . The aminoglycosides are generally ineffective against anaerobic organisms because the drugs require CQ-dependent, active transport into the bacteria (Verklin and Mandell, 1977). Pharmacokinetics. Aminoglycosides are poorly absorbed from the gastrointestinal tract, and because of their polarity, they poorly penetrate intracellular space and cerebrospinal fluid (Siegenthaler et al., 1986). They are 43 well tolerated following intramuscular (n: intravenous injection. Once absorbed, the aminoglycosides have a volume of distribution that is approximately that of the extracellular fluid compartment (Edwards et.al., 1981; Hall et al., 1977; Leroy et al., 1978; Schwartz et al., 1978; Walker et al., 1979). They are metabolically stable with 95% being eliminated via the kidneys. Antibacterial Activity. The antibacterial mechanism of action of the aminoglycosides is only partially understood. The aminoglycosides bind to the surface of the bacteria and are taken into the cell by an active transport mechanism (Bryan and Van den Elzen, 1977). Once inside the cell, they bind to the 308 ribosomal subunit, causing a misreading of messenger RNA during the translation process and producing "nonsense proteins" (Bryan, 1984; Moellering, 1983; Siegenthaler et al. , 1986) . Aminoglycosides do not dissociate once they bind to ribosomes. 'The irreversible binding results in a halt to cellular protein synthesis and an interference with cellular metabolism dependent on active protein synthesis sudh as membrane function. This causes potassium, sodium, amino acid, and other essential constituents to leak out, resulting in bacterial death. Nephrotoxic effects. The signs associated with aminoglycoside-induced nephrotoxicity are generally a rise in blood urea nitrogen (BUN) and serum creatinine (Davey and Harpur, 1987) , indicating a fall in glomerular filtration rate (GFR). This can proceed to non-oliguric renal failure. The 44 renal effects of aminoglycosides are generally reversible and seldom lead to a fatal outcome. .Although the clinical changes associated. with aminoglycoside-induced nephrotoxicity are those of decreased GFR, the renal toxicosis does not appear to involve the glomeruli: The primary area affected is the renal proximal (81-82 region) convoluted tubules (Houghton et al., 1976; Kosek et al., 1974). Aminoglycosides are eliminated unchanged in the glomerular filtrate and accumulate in the renal cortex at concentrations several times higher than that of the serum (Fabre et al., 1976; Giuliano et al., 1984). Most AA accumulate:in proximal tubular epithelial cells (Kuhar et al., 1979; Van de Walle et al., 1981), but small amounts are also reported to accumulate in patches in distal tubular epithelium (Bergeron et al., 1986; Silverblatt, 1982). The accumulation of the compounds is not by diffusion because they are too hydrophilic to freely cross cell membranes, but rather due to a process of absorptive endocytosis (Silverblatt and Kuehn, 1979). Once taken up, aminoglycosides are rapidly incorporated into lysosomes (Silverblatt and Kuehn, 1979). Further studies have shown that.before overt tubular necrosis, the: aminoglycosides are .almost, associated. with, lysosomes (Giurgea-Marion et al., 1986; Josepovitz et al., 1985). As aminoglycosides accumulate in the tubular epithelial cells, typical ultrastructural changes occur and consist of a progressive swelling of lysosomes (Houghton et al., 1976; Kosek et al., 1974). The lysosomal contents become 45 increasingly osmiophilic and lamellar in appearance (myeloid bodies). Enlarged lysosomes, many with lamellar profiles and electron-translucent centers, have been reported in cultured human skin fibroblasts after the addition.ofIgentamicin to the culture media (Oshima et al., 1986). In addition, a decrease in the rate of phospholipid turnover (phospholipidosis) in proximal tubular epithelium occurs (Ramsammy et al. , 1989a) in association with a marked reduction in lysosomal acid sphingomyelinase and phospholipase A1 activities (Feldman et al., 1982; Laurent et al., 1982; Tulkens, 1986; Tulkens and Van Hoof, 1980). The cytotoxicity of aminoglycosides has been attributed to mitochondrial dysfunction (Simmons et al. , 1980; Mela-Riker et al., 1986), perturbation of neoglucogenesis (Michalik and Bryla, 1987) , impairment of protein synthesis (Bennett et al. , 1988), and interactions with plasma membrane enzymes such as Na’, lU-ATPase (Williams et al., 1984) and phosphatidylinositol-specific phospholipase C (Schwertz et al., 1984). However, it is unclear whether these are a cause or a result of cellular dysfunction and/or necrosis. There exists good evidence of the relationship between the extent of tubular damage and the extent of inhibition of phospholipid catabolism (Toebeau et al., 1986). The close relationship between aminoglycoside-induced phospholipidosis and tubular necrosis is further demonstrated by the protective effects of anionic polypeptides against nephrotoxicity (Gilbert et al., 1989; Ramsammy et al., 1989b; Beauchamp et al., 1986) 46 The mechanism by which aminoglycosides cause inhibition of lysosomal phospholipase is not clear. Stearic hinderance resulting in a lesser accessibility to the substrate (i.e., phosphatidylcholine) has been proposed as one mechanism. This is supported by the association between aminoglycoside-induced inhibition Figure 8. Photomicrograph of the basilar papilla of chick embryos treated with gentamicin for 5 consecutive days with staining of gentamicin using the streptavidin-biotin method with DAB as the chromogen. A. Chick embryo, 0.1 mg/egg. Note diffuse staining of the basilar papilla. x 540. B. Chick embryo control. Note absence of staining of the basilar papilla. x 540 83 Figure 9. Photomicrographs of the basilar papilla of hatched chicks treated with gentamicin for 5 consecutive days with staining of with DAB as A. Hatched basilar B. Hatched diffuse gentamicin using the streptavidin-biotin method the chromogen. chick, 5 mg/kg. Notice diffuse staining of the papilla. x 540. chick, 100 mg/kg. Notice hair cell loss and staining of the basilar papilla and hair cell debris trapped in the tectorial membrane. x 540. C. Hatched chick control. Notice absence of staining of the basilar papilla. X 540. 84 Figure 9 85 transition from a tolerant to a sensitive state. A critical period of increased susceptibility to aminoglycoside ototoxicity during maturation of the cochlea has been shown to exist for guinea pigs (Uziel et al., 1979; Dumas and Charachon, 1982), rats (Osako et al., 1979; Carlier and Pujol, 1980; Lenoir et a1, 1983; Marot et al., 1980), cats (Bernard, 1981), and mice (Henry et al., 1981). In mammals, this period and.the onset and.development of auditory function are closely related (Lenoir et al., 1983). The onset of cochlear function coincides with the last anatomical stage of maturation and involves mainly final maturation of outer hair cells, which are generally regarded as the most sensitive to aminoglycoside antibiotics (Pujol et al., 1978; Lenoir et al., 1980). The existence of an insensitive phase prior to onset of the critical period is controversial. Studies performed in vivo indicate that the rat cochlea is insensitive to kanamycin treatment between birth and postnatal day 10, followed by the critical period beginning on day 11 (Osaka et al., 1979). The transition from an insensitive to sensitive period in postnatal mice is similar to that observed in this study as indicated by a rapid change from an insensitive chick embryo to a sensitive newly hatched chick. The chick critical period for the chick appears to be the first few days after hatching (Cruz et al., 1987; Hashino et al., 1991; Tucci and Rubel, 1990). .Studies examining the onset and.maturation.of hearing in the chick embryo and hatched chick indicate that a progressive maturation of auditory response occurs between the 86 fifteenth day of incubation and the first few days post- hatching (Rebillard and Rubel, 1981; Saunders et al., 1973). As in mammals, drug-induced hair cell loss in the chick during the first several days after hatching does coincide with the period of final development and maturation of function of the cochlea. The insensitivity of the chick embryo could be due to a lack of drug distribution to the basilar papilla. Indirect evidence of significant systemic distribution of gentamicin in the chick embryo after injection is denoted by renal tubular necrosis observed in chick embryos treated with gentamicin. This lesion was consistent with the nephrotoxic effects of aminoglycoside antibiotics (Laurent et al., 1990). In addition, immunocytochemical localization of gentamicin within the basilar papilla within 3 days indicates gentamicin is getting into the basilar papilla. It was not attempted in this study to relate the intensity of staining in the basilar papilla to an actual tissue concentration. If the basilar papilla of the chick embryo is accumulating gentamicin in concentrations comparable to those of the hatched chick from the high-dose group, then a lack of uptake into the hair cell or failure at a subsequent step in the mechanism of toxicity may be responsible for the insensitivity observed. Immunocytochemistry has been utilized in the localization of gentamicin and kanamycin in the cochlea (Tachibana et al., 1984; Veldman et al., 1987; Yamane et al., 1988; de Groot et al., 1990). Gentamicin was detected in the guinea pig cochlea 87 within 3-5 days (Veldman et al., 1987; de Groot et al., 1990). This interval closely matches that in chick embryos and hatched chicks treated with gentamicin in this study. The trend of gentamicin starting to accumulate in the basilar papilla as soon as 24 hr after treatment in all 3 treatment groups is similar to the rapid distribution of kanamycin to the organ of Corti following intravenous administration (Yamane et al,. 1988). Kanamycin reaches the organ of Corti within 10 min via capillaries of the basilar membrane and spiral sulcus, and within 90 min by penetration through the perilymphatic space» The ventral surface of the egg air space is lined by the chorioallantoic membrane. This membrane is highly vascularized and drugs injected into the air space and subsequently absorbed, may quickly distribute systemically via the circulatory system of the embryo. Specific labeling for gentamicin in the organ of Corti of guinea pigs included the outer and inner hair cells, as well as supporting cells such as Deiter's cells, Hensen's cells and the tympanic layer cells of the basilar membrane (de Groot et al., 1990. The pattern of gentamicin staining in this study certainly included supporting cells of the basilar papilla. Generally, hair cells did not appear to be labelled- To avoid interference with image analysis, tissue sections were not counterstained. This resulted in histologic detail not consistently allowing visualization of supporting cell borders. This precluded quantifying the lack of gentamicin staining in hair cells by image analysis. 88 The lack of morphologic alterations induced by aminoglycoside antibiotics in chick embryos of this study somewhat contrasts with the findings reported by Pickles et al. (1991). They reported a single dose of streptomycin injected in the center of an egg on days 7-11 of incubation resulted in 71% of embryos having hair cell morphologic changes. This compared to 18% affected on days 12-13 and none in those injected on days 14-15. Hair cells of chick embryos are clearly unambiguous by day 11 (Cohen and Fermin, 1978). Prior to this time, cytodifferentiation of the hair cell could be interfered with by a drug, resulting in a teratogenic (dysmorphogenic) effect rather than specific hair cell toxicity. Teratogenic effects have been produced in mouse cochlear explants exposed prior to the onset of morphogenesis on the 13th day of gestation (Anniko, 1981; Anniko and Nordemar, 1981). By 16 days of gestation, gross morphologic development has largely ended and exposure to drugs at this point resulted in selective hair cell toxicity. In the hatched chick, high-dose group, severe hair cell loss began after only 3 injections. Embryos were treated with gentamicin for 6 additional days after significant amounts of gentamicin were detected. in. the basilar' papilla. This suggests that if the chick embryos were within a period of sensitivity, adequate time was allowed for hair cell degeneration to be detected. 89 The chick embryo appears to be resistant to drug-induced ototoxicity and this resistance does not appear to be due to a lack of distribution of drug (gentamicin) to the cochlea. The following studies designed to examine the distribution of gentamicin within hair cells. CHAPTER 2 ULTRASTRUCTURAL IMMUNOCYTOCHEMICAL LOCALIZATION OF GENTAMICIN AND PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIPE) IN THE COCHLEA OF THE CHICK EMBRYO AND THE NEWLY HATCHED CHICK CHAPTER 2 ULTRASTRUCTURAL IMMUNOCYTOCHEMICAL LOCALIZATION OF GENTAMICIN AND PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIPZ) IN THE COCHLEA OF THE CHICK EMBRYO AND THE NEWLY HATCHED CHICK ABSTRACT To study the insensitivity of chick embryos to aminoglycoside-induced ototoxicity, the uptake of gentamicin by cochlear hair cells in chick embryos and hatched chicks was examined. Because the phospholipid phosphatidylinositol 4,5- bisphosphate (PIPZ) is critical in the mechanism of the ototoxicity, the intracellular distribution of gentamicin in relationship to PIPE in cochlear hair cells of chick embryos and hatched chicks was also examined. Chick embryos were dosed by air cell injection with gentamicin at 0.1 mg/egg/day during incubation days 10 - l8. Hatched chicks were dosed daily by subcutaneous injection with gentamicin at either 5.0 mg/kg (low-dose) or 100 mg/kg (high-dose) on post-hatching days 1 - 9. Ultrastructural immunocytochemical labeling for gentamicin and PIP2 in hair cells was performed 1 day after the lst, 3rd, 5th, 7th, and 9th doses of gentamicin. Mild, diffuse labeling for gentamicin was detected within endoplasmic reticulum of short and tall hair cells of chick 9O 91 embryos after 7 days (incubation day 17). Moderate labeling of gentamicin was detected in hair cell lysosomes after 5 treatments in chicks receiving gentamicin at 5.0 mg/kg and after 1 treatment in hatched chicks receiving gentamicin at 100 mg/kg. These lysosomes were consistently located in the infracuticular region” ‘There was an increase in the number of lysosomes with an occasional myeloid body coinciding with the accumulation of gentamicin within lysosomes in short and tall hair cells of chicks from both the low-dose and high-dose groups. Labeling for IUIQ was detected in all chick embryos and hatched chicks in groups at each time point. There was consistent labeling of PIP2 in the stereocilia, cuticular plate, chromatin, endoplasmic reticulum, cell walls, and tight junctions. Condensed PIP2 labeling in lysosomes was infrequently observed and little variation in the amount was noted in tissue from treated chick embryos or chicks regardless of treatment group or treatment day. In hatched chicks from the high-dose group, short and tall hair cell loss was detected after 3 doses of gentamicin, and degeneration of dark cells in the tegmentum vasculosum was observed after 5 doses. No ultrastructural evidence of hair cell loss, hair cell degeneration, accumulation of gentamicin within lysosomes, or an increase in the numbers of lysosomes was detected in hair cells from chick embryos. Since PIP2 and gentamicin were detected.in the hair cells from.chick.embryos, but there was no evidence of drug-induced ototoxicity, the intracellular concentration of gentamicin was either 92 insufficient to induce toxicity or a step in the mechanism of toxicity is uncompleted. INIBQDHQIIQN A period of insensitivity to aminoglycoside-induced ototoxicity has been reported in rats (Osako et al., 1979) and chick embryos (Fikes et al., 1992). This is followed by a sensitive period in both species (Osako et al., 1979; Carlier and Pujol, 1980; Cruz et al., 1987; Hashino et al., 1991; Park and Cohen, 1982) which coincides with the onset and.maturation of hearing (Alford and Ruben, 1963; Bosher and Warren, 1971; Rebillard and Rubel, 1981; Saunders et al., 1973; Shnerson et al., 1982). In the mechanism of aminoglycoside-induced ototoxicity by proposed Schacht and Weiner (1986), aminoglycosides are taken into the hair cell by an active uptake process followed by the critical step of interacting with the membrane phospholipid phosphatidylinositol 4,5- bisphosphate (PIPZ). Gentamicin has been reported to be present in the basilar papilla of chick embryos by embryonic day (ED) 17 when treatment started on ED 10 (Fikes et al., 1992), but it is unknown whether gentamicin is actually taken into the hair cells. Aminoglycoside-induced ototoxicity may occur i_n m in human beings (Scheinhorn and Angelillo, 1977) and animals (Akiyoshi, 1977), but if the mechanism of aminoglycoside tolerance observed in rat. pups and chick embryos was understood, then _i_n utero ototoxicity may be prevented. To determine the point at which the aminoglycoside 93 ototoxic mechanism of action may be uncompleted, the uptake of gentamicin into the hair cells of chick embryos and its intracellular distribution in relationship to PIPE ‘was studied. MATERIALS AND METHODS Fertile White Leghorn chicken eggs (Michigan State University, Poultry Research and Teaching Center, East Lansing, MI) were incubated (Petersime Incubator Model 5, Petersime Incubator Co., Gettysburg, OH) at 37°C at 50% humidity. After hatching, chicks were housed in brooders (Petersime Brood-Unit, Petersime Incubator Co., Gettysburg, OH) at 30°C. Chemicals were obtained as follows: gentamicin sulfate, G-3632, Sigma Chemical Co., St. Louis MO; 8% EM grade glutaraldehyde, Electron Microscopy Sciences, Fort Washington, PA; protein A-gold (10 and 20 nm label), E Y Laboratories, San Mateo, CA; goat whole antiserum to gentamicin, ICN Biochemicals, Costa Mesa, CA; and anti-PIP} :monoclonal antibody (Professor Tohru Yoshioka, Waseda University, Tokorosawa, Japan). Seventy-five fertile eggs were randomly divided into the following five groups: chick embryo, control group; chick embryo, treatment group; hatched chick, control group; hatched chick, low-dose group; and hatched chick, high-dose group. Chick embryos received either deionized water (0.1 ml) or gentamicin (0.1 mg/egg) via an injection into the egg air cell on incubation days U3-18. Hatched chicks were injected 94 subcutaneously beginning one day after hatching for eight consecutive days with deionized water (control group), gentamicin at 5.0 mg/kg (low-dose group), or gentamicin at 100 mg/kg (high-dose group). Three randomly selected eggs or chicks were removed from each group one day after the lst, 3rd, 5th, 7th, and 9th injections. Hatched chicks were anesthetized with CO2 before decapitation. The cochlea and.a section of kidney were removed and fixed as previously described (Fikes et al., 1992) except post-fixation with osmium tetroxide and uranyl acetate was omitted to avoid an adverse effect on antigenicity (Vardell and Polak, 1986). Tissues were dehydrated with ethanol, transferred to propylene oxide, and infiltrated with epon/araldite resin and embedded in the same resin. After curing for 60 hr at 60°C, a transverse thick section (1 pm), perpendicular to the longitudinal axis of the cochlea, was out along a plane approximately 30% of the total length from the base to apex of the basilar papilla. This section was used to assess cochlear'morphologyx This area should be central to that reported as showing hair cell loss in chicks treated.with aminoglycosides (Cruz et al., 1987; Hashino et al., 1991; Park and Cohen, 1982). Hair cell counts were completed by viewing each tissue section at a total magnification of x1000. Tall and short hair cells were combined for total hair cell count. The following criteria were used to include hair cells in the count: presence of a well-formed cell body, extension of the cell to the cuticular plate, and identifiable stereocilia 95 (Cruz et al., 1987). Three tissue sections/slide were counted and the total number of hair cells per tissue section averaged for the mean hair cell count for each slide. Three slides/group were then used to calculate the group mean and standard deviation. For ultrastructural immunocytochemistry, thin sections (70—90 nm) were cut and collected on thin-bar nickel grids. All sections were cut with a diamond knife on an LKBUUltrotome III (LKB-Produkter AB, Bromma, Sweden). Post-embedding immunocytochemistry (Roth et al., 1978; Bendayan, 1982; Bendayan et al., 1987) was performed by floating grids on droplets of immunoreagents and buffers placed on strips of Parafilm (American Can Co., Greenwich, CT). An indirect, four-step method was used to double label for PIPE and gentamicin simultaneously. In order to verify the somewhat arbitrary labeling'ofwgentamicin.in.chick.embryos with the double label technique, a single label method using a small (10 nm) label was used on the chick embryo tissue. In both techniques, 0.05M tris buffered saline (TBS) and 0.05% tween, 0.05M tris buffered saline (TTBS) were adjusted to pH 7.2 for use at the working temperature of either 4°C or 24°C (room temperature). For the indirect two-step method, grids were etched face down for' 5 min, with saturated sodium metaperiodate solution, rinsed with deionized water and pretreated for 30 min with 1% gelatin-TTBS. Each grid was transferred to a drop of gentamicin antiserum (1:450) in 1% BSA-TBS, for 16 hr at 4°C. After washing with TTBS, grids 96 were treated for 5 min with 1% gelatin-TTBS, followed by 30 min treatment with protein-A gold (10 nm) in TTBS. Each grid was rinsed with TTBS and deionized water. The sections were then stained with uranyl acetate and lead citrate and examined :h1 a Philips 301 transmission electron microscope (Philips Electronic Instruments Co., Pittsburg, PA). For double labeling, Eula was labeled first by floating grids face up for 5 min with saturated sodium metaperiodate solution to etch, rinsed with deionized water and pretreated for 30 min with 1% gelatin-TBS. Grids were then floated face up on a drop of anti-PIP2 monoclonal antibody (IgG3, 0.05 ug/ml) in 1% BSA-TBS for 24 hr at 4°C. After washing with TBS, grids were treated for 5 min with 1% gelatin-TBS, and for 30 min with protein-A gold (10 nm) in TBS. Each grid was rinsed with TBS and deionized water. For labeling gentamicin, the grids already labeled on the face up side for I015 were turned over and treated face down as described above for the indirect two-step method for gentamicin using a protein A-gold, 20 nm label, instead of 10 nm label. Following double labeling, sections were stained with uranyl acetate and lead citrate and examined. Sections of kidney from chicks from the high-dose group which were given 9 doses were used as positive controls for gentamicin (Figure 10). The specificity of the anti- gentamicin antibody following absorption with gentamicin is shown in Figure 11. Nonspecific binding of the antigentamicin antibody was quite low (Figure 12). 97 Figure 10. Transmission electron micrograph of a gentamicin immunolabeled section of kidney from a 10-day-old hatched chick after 9 days of treatment with gentamicin at a dose of 100 mg/kg. Labeling for gentamicin is restricted to lysosomes (L) and rough endoplasmic reticulum (RER). x 65,000. 98 Figure 11. Transmission electron micrograph of a gentamicin immunolabeled section of kidney from a lo-day-old hatched chick after 9 days of treatment with gentamicin at a dose of 100 mg/kg following preincubation of anti-gentamicin serum with gentamicin. Same section as Figure 10. Note absence of staining of lysosomes (L), rough endoplasmic reticulum (RER). x 65,000. 99 Figure 12. Transmission electron micrograph of a gentamicin immunolabeled section of kidney from a 10-day-old hatched chick, negative control, after' 9 days of treatment with deionized water at 0.1 ml/day. No staining of lysosomes (L) and rough endoplasmic reticulum (RER). x 65,000. 100 The specificity of labeling with anti-PIPE antibody was demonstrated by an absorption test with inositol 1- monophosphate (IPQ, inositol 1,4-bisphosphate (1R9, and inositol 1,4,5-triphosphate (IRQ. Since the epitopes of the anti-PIPQ antibody are phosphate groups substituted at the 4- and 5-positions of the inositol head of PIP2 (Miyazawa et a1. , 1988), 113 can act as an absorber for this antibody (Ito et al., 1991). Labeling of a negative control chick hair cell cuticular plate and stereocilia is demonstrated in Figure 13. When anti-PIPZ antibody was absorbed with an excess of 1P3, immunolabeling was markedly abolished (Figures 14, 15). Preincubation of anti-PIP2 antibody with IP1 and IP2 had no effect on labeling. BE§QLI§ Labeling for gentamicin with the double labeling technique was negative through the first 5 days of treatment and equivocal after 7 and 9 days (Figure 16), but using the single labeling technique, gentamicin was detected in small amounts within short and tall hair cells of chick embryo cochlea after 7 injections (Figure 17). In hatched chicks from the low—dose group, there was an accumulation of gentamicin label within lysosomes which was detected after 5 injections (Figure 18). In the tissues from chicks in the high-dose gentamicin group, gentamicin was detected within lysosomes after one treatment (Figure 19). Gentamicin was rarely detected in mitochondria, golgi, or endoplasmic Figure 13. Transmission electron micrograph of a PIP immunolabeled section of a tall hair cell from a 6-day-ol hatched chick, negative control, cochlea after 5 days of treatment with 0.1 ml deionized water/day. Labeling for PIPZ is restricted to stereocilia and cuticular plate (CP). x 88,400. 102 .56“: 3 . Haw Z A-* '1... v ,3 m 4}" ' )- / I 5 ..MA Q. Figure 14. Transmission electron micrograph of a PIP2 immunolabeled section of tall hair cell stereocilia from a 6- day-old hatched chick, negative control cochlea after 5 days of treatment with 0.1 ml deionized water/day following preincubation of anti- -PIP2 antibody with an excess of IP. Same section as Figure 13. 3 Note marked elimination of labeling of stereocilia. x 88 400. 103 Figure 15. Transmission electron micrograph of a PIP2 immunolabeled section of a tall hair cell cuticular plate (CP) from a 6-day-old hatched chick, negative control, cochlea after 5 days of treatment with 0.1 ml deionized water/day following preincubation of anti-PIP2 antibody with an excess of IP . Same section as 13. Note marked elimination of labeling of cuticular plate. x 88,400. 104 Figure 16. Transmission electron micrograph immunolabeled section of a short hair cell from a on incubation day 17 after 7 days of gentamicin a dose of 0.1 mg/egg. Notice diffuse labeling small label (10 nm), only equivocal labeling gentamicin with large label (20 nm). x 65,000. of a double chick embryo treatment at of PIP2 with (arrows) of 105 Figure 17. Transmission electron micrograph of a gentamicin immunolabeled section of a short hair cell from a chick embryo on day 17 of incubation after 7 days of gentamicin treatment at a dose of 0.1 mg/egg. Label (10 nm) for gentamicin in areas of rough endoplasmic reticulum (asterisk). x 88,400. 106 Figure 18. Transmission electron micrograph of a double immunolabeled section of a tall hair cell from a 6-day-old hatched chick after 5 days of gentamicin at 5.0 mg/kg. PIP2 stained with small label (10 nm) and gentamicin stained with large label (20 nm). Note gentamicin staining within lysosomes (arrows). Diffuse PIP2 staining (arrowheads) over cytoplasm. x 65,000. 107 Figure 19. Transmission electron micrograph of a double immunolabeled section of a short hair cell from a 2-day-old hatched chick after 1 day of gentamicin treatment at 100 mg/kg. PIPZ stained with small label (10 nm). Gentamicin stained with large label (20 nm) . Note gentamicin accumulated within lysosomes (arrows). Some labeling of PIP2 within lysosomes. x 65,000. 108 reticulum of the hair cells. Label for anti-PIPa was detected in hair cells from chick embryos and from.hatched chicks (Figures 17, 18, 19). In.hair cells of chick embryos, PIP2 was present in stereocilia, cuticular plate, nucleus, endoplasmic reticulum, cell wall and tight junctions. PIP2 labeling within lysosomes was variable. Occasionally the PIP} labeling was concentrated (Figure 20). The distribution of PIP2 within hair cells of chicks from the low- and high—dose gentamicin groups was essentially the same as described for the chick embryos. Hair cell loss was detected in the hatched chick, high- dose group after three injections of gentamicin (Table 3). Once hair cell loss occurred, it did not appear to increase in severity over the remainder of the study. Both short and tall hair cells were lost, frequently leaving only an occasional hair cell (Figure 21). Extruded hair cells and cellular debris were located within and beneath the tectorial membrane (Figure 22). Intranuclear pseudoinclusions characterized by margination of chromatin and condensation of chromatin into focal aggregates were present in dark cells of the tegmentum vasculosum of chicks after 5 injections (Figure 23). This degenerative change was progressive and karyolysis was detected in chicks after 9 injections. INo histologic evidence of hair cell degeneration or loss was detected in chick embryos from the control or treatment groups or in the hatched chicks from the control or low-dose groups. 109 Transmission electron micrograph of ii double Figure 20. and Labeling for Focal area of labeling gentam1c1n ls present w1th1n lysosome. for PIP2 within lysosome (arrow). x 65,000. -day-old (10 nm) stained with small label h large label (20 nm) 2. w1t hatched chick after 7 days of gentamicin treatment at a dose PIP immunolabeled section of a short hair cell from an 8 of 100 mg/kg. gentamicin stained 110 .>Mo\umu03 UmuHcono HE H.o .>mo\ox\cH0HEmucwm ma ooa M .>0o\mx\cHoHEmucmm ma o.m u .>mo\mvm\uwum3 UmNHcono HE H.o u .mmo\mmm\cHoHEmucmm me H.o n mooum\mwuna a N.N H ¢o.¢a m.m H 0N.H m.o H mm.ma h.H H mm.NH m.H H oo.mH m m.H H bm.¢H v.0 H ha.m ¢.H H NN.NH m.o H om.MH H.H H om.NH b 5.0 H hm.ma ~.H H hm.a o.m H oo.ma m.H H mm.NH m.H H mm.ma m v.H H Hh.NH m.m H hm.m m.o H mm.va H.H H ¢N.ma 0.0 H no.ma m H.H H wm.ma o.H H mw.ma N.H H hm.ma m.o H wb.ma m.o H oo.MH H Houucoot mmoolcmHmo wmool3oqn Houucoou chfiuwmupa cHoHampcmO x020 nmnoumm x030 cmnoumm 0830 8028.81 0305 x030 0505 0830 mo mama .CHOHEmucmv «~qu omumwuu monno omnoumca 0cm moxnhnfim 03.20.. CH mocmumHo xwmm ou mmon mo won an unsoo Hamo “Hon Homacooo AcoHumH>mo Unmocmum Hy :00: .m mHQwB Figure 21. 111 Photomicrograph of a section of basilar papilla from a 4—day-old hatched chick treated with gentamicin at 100 mg/kg for 3 days. (arrows). Notice loss of short and tall hair cells Toluidine blue. x 830. 112 A. ' y.- . V I ~, . - a. .‘_"'. " q " - ~ ‘. ' : ,1 . , .. . , .y , _ I ‘. - . - . 1 u h I ' ’ I. . . ‘ i .0 ' ' I _ a Q” J" V. _5 Figure 22. Transmission electron micrograph of a section of basilar papilla from a 4-day-old hatched chick treated with gentamicin at 100 mg/kg for 3 days . Basilar papilla (BP) and tectorial membrane (TM). Extruded, degenerating hair cells (arrows) trapped in the tectorial membrane. x 6,500. 113 O r I 1. .0 a r ' . 6" 4” 0 to) * tilt ’ ~ ‘ "O x .’ ). .D W a ” .4“ ‘3 a ':_C' "'O 0 - ' Figure 23. Photomicrograph of a section of tegmentum vasculosum from an 8—day-old hatched chick cochlea after gentamicin at 100 mg/kg for 7 days. Note pyknotic nuclei (arrowheads) and pseudoinclusion formation (arrow) in dark cells. Note adjacent normal appearing light cells. Toluidine blue. x 736. DISCUSSION The results of labeling gentamicin in the cochlea of chick embryos after 7 injections indicates some gentamicin is being taken into the cell. It was located in endoplasmic reticulum and other ribosomal dense areas which is consistent with the hypothesized intracellular movement of gentamicin before being concentrated into lysosomes (de Groot et al., 1990). The accumulation of gentamicin within lysosomes of hair cells in this study is similar to the rapid incorporation into lysosomes seen in the kidney (Silverblatt and Kuehn, 1979). The accumulation of gentamicin within lysosomes occurred in conjunction with an increase in number of lysosomes and the formation of myeloid bodies. Not every lysosome labeled for gentamicin. This could be due to an increase in number of lysosomes as a cellular response to the toxic effects of gentamicin and not simply a mechanism to sequester the drug. The increase in the number of lysosomes is attributed to a drug-induced block in lysosomal phospholipid catabolism with accumulation of phospholipids (phospholipidosis) within lysosomes (Laurent et al., 1990). The increased number of lysosomes observed in hair cells were primarily restricted to the infracuticular region" Gentamicin-labeled lysosomes were always in this area, consistent with the findings of de Groot et al. (1990). In the hatched chick groups, labeling of gentamicin in lysosomes was generally equal between short and tall hair cells. Distribution oflgentamicin to both inner and 114 115 outer hair cells of guinea pigs has also been reported (Hayashida, 1989). This suggests that a differential uptake between hair cell types does not account for previously observed increased sensitivity of outer hair cells. Studies have shown. that before overt renal tubular necrosis, the aminoglycosides are almost exclusively associated with lysosomes (Josepovitz et al., 1985; Giurgea- Marion et al., 1986), which act as the major intracellular storage site for gentamicin in spite of numerous changes in cell homeostasis (Giurgea-Marion et al, 1986). Although both hatched chick groups demonstrated ultrastructural evidence of gentamicin accumulation in lysosomes and phospholipidosis, only the high-dose group had hair cell loss. This may be explained by the premise that a critical threshold of phospholipid accumulation needs to be reached before it leads to cell death (Giuliano et al., 1984). This is supported in this study by the subjective observation of a larger number of lysosomes appearing over time in the hair cells of chicks from the high-dose group than those in the hair cells of chicks from the low-dose group. Although many phospholipids increase in conjunction with the phospholipidosis, apparently PIP‘2 does not change during the early manifestations of nephrotoxicity (Feldman et al., 1982). Since focal increased labeling of lysosomes or the presence of myeloid bodies with anti--PIP2 was rarely observed in this study, PIP2 apparently does not change during the early manifestations of ototoxicity in hatched chicks. 116 Phosphatidylinositol 4,5-bisphosphate (PIP3)‘was present in the youngest chick embryos of this study and was probably not a limiting factor in the production of ototoxic effects in the chick embryos“ 'The similar labeling intensity of cochleas between chick embryos and hatched chicks is consistent with the finding that the turnover of PIPé‘within the cochlea is similar in fetal rats as adults (Anniko and Schacht, 1981). Hair cell loss after 3 injections in the hatched chicks from the high-dose group was slightly earlier than the 8-10 days reported in several studies (Cruz et al., 1987); Lippe, 1991). This could be due to the higher doses of gentamicin (100 mg/kg) used in this study, in contrast to gentamicin at 50 mg/kg/day used by Cruz et al. (1987). This study also included an earlier time point than most studies. The differences observed between the single and double labeling techniques for gentamicin in chick embryos of this study indicates the immunocytochemical techniques used may have limited sensitivity. The detection of gentamicin in the endoplasmic reticulum of hair cells by immunocytochemical methods was rare (de Groot et al., 1990) and was attributed to drug concentrations being below that which could be detected by the method. In the present study, the differences in sensitivity may be due to the different sizes of labels used to detect gentamicin in the two techniques. Smaller gold particles allow higher labeling densities and improved sensitivity (Horisberger, 1981; Lackie et al., 1985). The higher labeling density results from the greater accessibility 117 of the smaller particles to binding sites and lower steric interference as compared to larger gold particles. larger particles give a lower staining density than smaller ones because of steric hinderance, lower mobility of large particles, and higher mutual electric repulsion of large particles (Doerr-Schott, 1989, Park and Park, 1989). From the present study, it appears that gentamicin is getting into the hair cells of chick embryos near the end of the dosing schedule, but there was no evidence of ototoxicity, such as hair cell loss, increase in lysosome numbers, or accumulation of gentamicin in lysosomes. This raises several possibilities for explaining the insensitivity of the chick embryo to aminoglycoside-induced ototoxicity. Gentamicin uptake into hair cells had only begun 2 days prior to the end of this study indicating that the energy-dependent uptake mechanisms may not yet be fully functional. A second possibility is that once gentamicin is taken into the cell, bioactivation of gentamicin may not occur due to oxidative enzyme immaturity. It is generally regarded that the aminoglycosides are not metabolized (Laurent et al., 1990), and are therefore nephrotoxic and ototoxic as the parent compound. This tenet, however, may be incorrect in that recently, an oxidative metabolite was shown to be cytotoxic (Huang and Schacht, 1990). .As a third.possibility, gentamicin may be taken into the hair cell, but fail to complex with PIPZ. 118 In summary, this study demonstrated the presence of gentamicin within hair cells of chick embryos by day 17 of incubation, following 7 days of treatment. This was later than that of hatched chicks and no accumulation. within lysosomes was detected. By day 11 of incubation, PIP2 was present in the hair cells of chick embryos, suggesting it may not be the limiting factor in the chick embryo's insensitivity to aminoglycosides. However, the interaction of gentamicin with PIPE once it was taken into the cell still needs to be determined. CONCLUSIONS AND FUTURE STUDIES CONCLUSIONS AND FUTURE STUDIES These studies were originally initiated in an attempt to use the chick embryo in screening tests for ototoxic compounds. Support for this hypothesis came from studies utilizing chick embryos in alternative "i_11 Lit—rg" ocular irritation tests instead of using rabbits in the traditional Draize test (Leighton et al., 1985) and studies demonstrating aminoglycoside antibiotics produce cochlear and vestibular hair cell loss in newly hatched chicks (Cruz et al,. 1987; Duckert and Rubel, 1990; Hashino and Sokabe, 1989; Hashino et al., 1991; Park and Cohen, 1982; Tucci and Rubel, 1990). During preliminary studies, it became apparent that chick embryos were tolerant to aminoglycoside-induced ototoxicity. with.this background, the present research was directed.toward verifying the insensitivity of the chick embryo to drug- induced ototoxicity and to begin a systematic characterization to understand this phenomenon. The chick embryo is not a good animal to use in a screen for ototoxic compounds because of this insensitivity to the aminoglycosides and a loop diuretic (ethacrynic acid). This conclusion is based on the following three results described 119 120 in Chapters 1 and 2: (1) Gentamicin is taken into the hair cell of chick embryos late in the treatment period (ED 17). The intensity of gentamicin labeling within short and tall hair cells was light and dispersed within areas of endoplasmic reticulum. (2) No ultrastructural or histologic evidence of hair cell loss is detected in chick embryos treated with aminoglycoside antibiotics or aminoglycosides potentiated with a loop diuretic. (3) No ultrastructural evidence of aminoglycoside-induced phospholipidosis was observed in chick embryo hair cells. Additional noteworthy results described in Chapters 1 and 2 include: (4) In hatched chicks, cochlear hair cell loss began after 3 treatments with gentamicin at 100 mg/kg. (5) In hatched chicks treated with nontoxic and ototoxic doses of gentamicin, gentamicin labelling appeared as focal accumulations within lysosomes in the infracuticular region of short and tall hair cells, An increase in total number of lysosomes, both with and without gentamicin label, occurs in the infracuticular region. (6) Gentamicin is distributed to the basilar papilla of the chick embryo after 3 days of treatment (ED 13). The rate of gentamicin appearance and its pattern of distribution in the basilar papilla of the chick embryo is similar to that of hatched chicks exposed to ototoxic doses of gentamicin. 121 This research has provided fundamental information regarding the basis of the insensitivity of chick embryos to ototoxicants, but three particular areas of research should be pursued. First, quantitation over time of aminoglycoside drug (i.e. gentamicin) concentration in the basilar papilla in chick embryos compared to the quantitation over time of concentration in the basilar papilla in hatch chicks treated with ototoxic doses of drug would provide some direct information on the pharmacokinetics of drug movement in the chick embryo cochlea. Although gentamicin is distributed to the basilar papilla of chick embryos early in the dosing period (ED 13), the amount relative to that delivered to the basilar papilla of chicks receiving ototoxic doses.is unknown. In the present studies, the IODs of the basilar papilla were similar between chick embryos treated with gentamicin at 0.1 mg/egg and hatched chicks treated.with.gentamicin.at 51and 100 mg/kg. A direct correlation between staining intensity and drug concentration would indicate that the concentration of gentamicin is similar in the basilar papilla of all three of these treatment groups. This also suggests that delivery of an aminoglycoside to the basilar papilla has little correlation with subsequent ototoxicity. Determination of this would provide some direct information on the pharmacokinetics of drug movement in the chick embryo cochlea. Although the basilar papilla of the chick embryo stained for gentamicin, the actual concentration in the structure may not 122 be high enough to serve as a pool for delivery to the hair cells. A second area of research is to more clearly define the "width" of the window of transformation from an ototoxicant- tolerant state to a sensitive state. This could be investigated by sequentially moving the dosing period forward from one day after hatching into the embryonic period. The present work identified a tolerant state, but did not establish an incremental conversion to the sensitive state. If an incremental process is identified, later studies could be concentrated on exactly what unique events are occurring within that specific time frame. A third area of future research is the examination of concentrations of gentamicin within isolated hair cells from 1 vivo treated chick embryos and hatched chicks. The present study demonstrated that hair cells from chick embryos began taking up gentamicin late in the treatment period (ED 17). However, the relevance of the amount identified is unknown. Comparison over time of drug concentration in the hair cells of chick embryos to that in hatched chicks given an ototoxic dose would provide some indication of the pharmacodynamics at the cellular level. Aminoglycosides may enter into the hair cell apical surface from the endolymph or the path of aminoglycosides into the hair cell may involve supporting cells, which may be a limiting factor. Regardless of the route of drug passage to the hair cell, the entire insensitive state in the chick embryo could be due to insufficient drug 123 uptake by a transiently immature transport mechanism, preventing intracellular cytotoxic accumulations. Also, more general questions to consider in planning future studies include: are aminoglycoside insensitive hair cells also insensitive to noise-induced trauma and can hair cells sensitive to aminoglycosides be made insensitive? Before pursuing these questions, a better understanding of the mechanism of insensitivity would be helpful. However, concurrent investigation of the insensitivity to aminoglycoside-induced ototoxicity and these general issues may simultaneously provide information on the mechanism of the insensitivity and application of the knowledge in a more general situation. Understanding the insensitivity of the chick embryo to aminoglycoside-induced ototoxicity is important from the aspect of what it offers in decreasing the incidence of this therapy limiting side effect. Also, since ototoxicity can be the limiting factor in the use of aminoglycosides, a means to avoid this may allow enhanced use of aminoglycosides in critical infectious disease cases. Although the chick embryo is not a good model for screening for ototoxicity, the newly hatched chick does make a suitable model. At one to two weeks of age, the chick inner ear is still easily'dissectedn Literature currently exists on the sensitivity of chicks to aminoglycoside-induced ototoxicity and noise-induced trauma which supports the concept of using chicks as a screening test for ototoxicants. 124 This system. would require further ‘validation. with other ototoxicants such as loop diuretics (ethacrynic acid), antineoplastic agents (cisplatin), nonsteroidal antiinflammatory drugs (aspirin), and antimalarials (quinine). The effects of these drugs in human beings is well characterized and similar results would be desired in the chick if it were to be used in an ototoxicity screen. Use of the adult chicken in otologic research is rarely reported in the literature. Although adult chickens may have similar sensitivities as young chicks to ototoxicants, the increased logistics of maintaining adult chickens and increased difficulty in dissecting and processing the inner ear tissue make it unlikely that adult chickens would be advantageous over chicks as an ototoxicity screen. APPENDIX APPENDIX INTRODUCTION Information regarding the toxicity of aminoglycoside antibiotics and loop diuretics is generally unavailable. It was therefore necessary to perfomnla series of studies to determine the lethal and maximum tolerated doses of the drugs using chick embryos for the studies described in Chapters 1 and 2. MATERIALS AND METHODS Fertile White Leghorn chicken eggs (Michigan State University, Poultry' Research. and 'Teaching’ Center, East Lansing; MI) were incubated (Petersime Incubator Model 5, Petersime Incubator Co., Gettysburg, OH) at 37°C with 90% humidity. Drugs were obtained as follows: gentamicin sulfate (G-3632, Sigma Chemical Co., St. Louis, MO); streptomycin sulfate (S-6501, Sigma Chemical Co., St. Louis, MO); kanamycin sulfate (Kantrim, 200 mg/ml, Fort Dodge Laboratories, Inc., Fort Dodge, IA); ethacrynate sodium (Sodium Edecrin, 50mg, Merck, Sharp, and Dohme, West Point, PA). Fertile eggs were exposed to the aminoglycoside antibiotics gentamicin, kanamycin, and streptomycin and/or the loop diuretic, ethacrynic acid. Drugs were administered by the routes, time periods, and dose ranges listed in Table 4. For each trial, eggs were candled one to two times per day to assess embryo viability. 125 126 H8838 me HO.O u HOO.O + >mo\mmm\unfim MOHSO CH mmHUSHm mmcommmu .coHumuuchHEUm mo mmuoom .v manna 127 Air cell injection was performed by candling the egg in a perpendicular plane and marking the limits of the air cell with a pencil. A mark was made at.a point approximately equal distance from the limits of the air space. The area around the mark was disinfected with 70% ethanol and allowed to dry. A small hole was drilled through the shell at the pencil mark, but not through the shell membrane. The area around the hole was disinfected with 70% ethanol and allowed to dry. Eggs were injected by holding them in a perpendicular fashion and inserting a 25 gauge, 5/8th inch needle, attached to a 1 ml tuberculin syringe, approximately 1/4th of an inch into the hole. Following injection, the hole was sealed with melted paraffin. On subsequent injections, the egg was held in a perpendicular plane and the paraffin cap and surrounding area disinfected with 70% ethanol. The paraffin cap was scraped free and the ethanol allowed to dry. The egg was then injected and resealed as previously described. For allantoic cavity injections, eggs were candled in a perpendicular plane and the limits of the air cell was outlined in pencil. A mark was made approximately 1/8 inch above the line over the air cell on the side opposite the developing embryo. The area around the mark was disinfected with 70% ethanol and allowed to dryu A small hole was drilled through the shell at the pencil mark, but not through the shell membrane. 'The area around the hole was disinfected.with 70% ethanol and allowed to dry. Eggs were injected by holding them in a perpendicular plane and, using a 1 ml tuberculin 128 syringe fitted with a 25 gauge 5/8 inch needle, inserting the needle perpendicularly through.the hole to the hub. Following injection, the hole was sealed with melted paraffin. On subsequent injections, the egg was held in a perpendicular plane and the paraffin cap and surrounding area disinfected with 70% ethanol. The paraffin cap was scraped free and the ethanol allowed to dry. The egg was then injected and resealed as previously described. For yolk sac injections, eggs were candled with the long axis in the horizontal plane to locate the yolk sac. A pencil mark was made on the shell over the yolk sac about half-way from the small end of the egg to the apex of the curvature of the shell. The area was disinfected by applying 70% ethanol and allowed to dry. A small hole was drilled through the shell but not through the shell membrane. The area around the hole was disinfected with 70% ethanol and allowed to dry. Injection.was performed by holding the long axis of the egg in the horizontal plane and using a 1 ml tuberculin syringe fitted with a 25 gauge 5/8 inch needle, the needle was passed perpendicularly through the hole to the hub RESULTS Gentamicin Studies Air cell injection of gentamicin at a dose between 5 to 40 mg/egg/day produced death of chick embryos after 2 treatments of the highest dose group after 2 treatments and after 6 treatments of the lowest dose (Table 5). No chick 129 embryo lived longer than ED 18. Air cell injection of gentamicin at a dose between 0.01 mg to 1.0 mg/egg/day caused a dose related mortality. Administration of gentamicin at a dose of 1.0 mg/egg/day resulted in chick embryo death beginning on ED 14 (Table 6). Gentamicin at a dose of 0.5 mg/egg/day produced chick embryo death beginning on ED 17 and at a dose of 0.1 mg/egg/day caused chick embryo death beginning on ED 19. Death of chicks receiving gentamicin at a dose of 0.01 or 0.05 mg/egg/day was infrequent. Gentamicin air cell injection during ED 10-14 or ED 15-19 at a dose between 0.01-1.0 mg/kg/egg was performed to compare the effect of injection early in incubation (ED 10-14) and late in incubation (ED 15-19) on mortality. Although chick embryo death was noted during both time periods, the time of injection had little effect on mortality (Table 7). Allantoic space injection of gentamicin also produced chick embryo death. Chick embryos given gentamicin at a dose of 5.0 mg/egg/day in allantoic space starting on ED 10 began dying by ED 18. Doses of gentamicin between 1.0 to 4.0 mg/egg/day caused death between ED 18-20 (Table 8). Yolk sac injection of gentamicin at a dose of 5.0 mg/egg/day caused chick embryo death beginning on ED 16 with no chick embryos surviving past ED 17 (Table 9). Administration of gentamicin at a dose of 3.0 mg/egg/day was lethal beginning on ED 17 with no chick embryos surviving past ED 19. A single chick embryo receiving gentamicin at a dose of 1.0 mg/egg/day died on ED 19. 130 .mmm\mfi ov .mmm\me om .mmm\qfi om .omm\mE mH .mqm\mfi oH .qom\ofi m .mmm\uwums omNHCono HE H.o .uumum um momunfiw onC0\mCHCHmEmu mo>HQEm onCo 0>HHH "0 macaw "m msouo um macho "a QDOHU "O moono “m macho "C macho m\O m\~ m\m m\m o mxo m\H m\m m\m m m\O m\~ m\~ m\m m\m m O\O m\H m\~ m\~ m\m m\m m\m a m\O m\H m\m m\m m\m m\m o m\O m\m m\~ m\m m\m mxm m\m m\m mxm m ~\~ m\~ ~\m ~\m mxm ~\~ m\~ ~\~ m\m, a OH RH OH OH OH HH NH HH OH QSOHU >00 CoHHMQCOCH Cqu HHwo HHm OCH CUCH omuownCH mmmm meoHCO 60u0C0>Hn50 CH wuHHmuHoz .RH Cmdounu OH mwmo CoHHMQCOCH Co wmo\mmm\ofi o¢ ou o.m um CHoHEmqum .m OHQMB 131 .qom\me .mqm\mfi .mqm\mE .mmm\ma .mmm\mE oo.H om.o oa.o mo.o Ho.o .mom\um#03 CONHCono HE H.o .uumum um mo>unfim xOHC0\mCHCHmEmH mowunfim xOHCO m>HHF "h "M "Q um u< macho QCOHO macho macho QCOHO QCOHO O\O O\H o\m O\m O\O O\O O\O O\O m O\H O\H O\m O\m O\O O\O m\m O\O O\O o\O m R\m R\O R\O R\O R\O R\R R\R R\R R\R R\R o R\O R\O R\O R\O R\O R\O R\O R\O R\O R\R o R\O R\R R\R R\R R\R R\R R\R R\R R\R R\R m R\m R\m R\m R\m R\m R\m R\m R\O R\O R\RF H OH OH RH OH OH 4H mH NH HH OH QCOHO zoo CoHUMQCOCH .RH Cmsounu 0H mxmo COHumnCOCH Co >mo\0mw\ma o.H ou Ho.o um CHOHEmqum CuHs woman HHm 0:» CH omuomflCH mmmm meoHCo 0090C0>H950 CH thHmuuoz .0 OHQGB 132 .oom\CHoHEmHCwm ma o.H .mmm\CHoHEmpCmq ma m.o .mmm\CH0HEmqum ma H.o .mom\CHoHEmqum we mo.o .mmm\CHoHEmquo OE Ho.o .@00\CHOHEqumm ma m.o .mmm\CHOHEmHCmm ma H.o .mmm\CHoHEqumo ma mo.o .omm\CHOHEmqum we Ho.o .uuoum um moxhnam xOHCo\mCHCHmEmH mo>HnEm xOHCo m>HHF “H macho “m msouw no QCOHO "m QCOHO um QCOHO "a QCOHU “O msouw "m QCOHO “C QCon O\O O\~ O\O O\O «\O O\O O\O O\O O\O H O\O O\~ O\O O\O O\O O\O O\O O\O O\O O\O m O\H OxO O\O O\O O\O O\O O\O O\O O\O O\O O\O O O\O O\O O\O O\O O\O O\O O\O O\O O\O O\O O\O O mHImH w>mo OHComnnfim mCHHCo ommoo O\O O\O «\O OxO «\O O\O O\O O\O «\O O\O O\O m O\O O\H Oxm O\m O\O O\O O\O O\O O\O O\O O\O O O\~ O\O O\O O\O O\O O\O O\O O\O O\O O\O O\O o O\O O\O O\O O\O O\O O\O O\O O\O O\O O\O O\O m O\O O\O O\O O\O O\O O\O O\O O\O O\O O\O O\O. O OHIOH m>mo OHC0>HQE0 mCHHCo ommoo ON OH OH RH OH OH OH OH NH HH OH QCOHO >00 CoHquCOCH .mH Cmsounu mH m>mo Ho OH Cqsounu 0H mzmo CoHumnCOCH Co CHOHEOqum CuHS woman HHm OCH CH omuomnCH womm CoxOHCO owumCo>HnEw CH wuHHmuHoz .h OHQMB 133 .mmm\CHOHEOquq we .mom\CHoHEOqum we .mmm\CH0HE0quu m5 .mom\CHOHE0qum ma .@@0\CHOHE0quo ma .uu0um H0 moxunfim onC0\mCHCH0Emu mo>unfim m "m QCOHO O .o msouw m “U msouw m Hm QCOHO H "a macho xOHCo 0>HHP O\O O\O O\O O\O O\m «\O O\O O\O O\O m O\O mxH m\~ m\m O\O O\m O\m m\m m\m O\O O\O O O\O O\H O\O O\O O\m «\O O\O «\O O\O O\O o O\O O\H O\m «\O «\O O\O O\O «\O «\O «\O «\O O\O m O\H O\H O\O O\O «\O O\O O\O O\O O\O O\O O\O «\O, 0 HN om mH mH OH OH mH vH MH NH HH OH QCoHO >0Q C0HH0QCOCH .RH CmsouCu 0H m>0o CoHu0QCOCH Co 000mm oHouCOHHO 0C» oHCH CHOHE0qumO CuH3 omuothH moom waOHCO omu0Coxunfim CH .>HHHOu.Hoz .m 0.308 134 .mom\CHoHE0qum 0E m "U QCOHO .mmm\CHoHE0qum 05 m "0 msouw .mmm\CHoHE0uC00 me H "0 QCOHO .uu0um #0 mo>HQE0 xOHCO\0CHCH0EmH mo>unew xOHCo 0>HHH O\O O\~ O\O O\O O\O OxO OxO O\O «\O o O\O O\m O\m O\m O\O O\O Oxm O\m O\m O\m O\O m Oxm Oxm O\~ O\O mxm O\O O\O O\O O\O m\m O\m O\O O\OF 0 H+m Hm Om OH OH RH OH OH OH OH OH HH OH macho >00 CoHu0QCOCH .RH CmCoHCv 0H m>00 CoHp0QCOCH Co 000 xHo> OCH opCH CHUHE0HC00 Csz cwuownCH 0000 meoHCo 00u0Co>Hnfim CH >uHHOuuoz .O 0HQOB 135 Chick embryos were also exposed by submersion of the egg in solutions of gentamicin. Because of the temperature differential between the warm egg and the cold water, solutions are readily taken into the egg. In order to determine what temperature of solution would result in an adequate amount of solution uptake by the egg, a trial using 1.0% gentamicin solutions maintained at 4.5-6.0°C, 11-16°C, or 20-21°C was completed. Three eggs/group were weighed every two minutes during the submersion and the cumulative weight gain recorded (Table 10). Submersion in the gentamicin solution at 4.5-6.0°C for 10 minutes resulted in a cumulative gain of 0.405 t 0.034 grams while submersion for 10 minutes at 11—16°C or 20-21°C resulted in a cumulative weight gain of 0.101 t 0.021 grams and 0.191 t .001 grams, respectively. In a second submersion trial, eggs were submerged for 10 minutes in 5-7°C solutions of deionized water, or gentamicin at 2.5, 12.5 , or 25 mg/ml. Mortality occurred after 1 day of treatment in all groups including the deionized water group, with no chick embryo living past ED 16 (Table 11). Kanamycin Study Air cell injection of kanamycin beginning on ED 10 was lethal in all chick embryos receiving 100 mg/egg/day after one treatment and in chick embryos receiving 10 mg/egg/day after two treatments (Table 12). Deaths began to occur on ED 17 in chick embryos receiving kanamycin at a dose of 1.0 mg/egg/day while no deaths occurred in chick embryos receiving doses between 0.01 or 0.1 mg of kanamycin/egg/day. 136 .O.Hmlom H0 00CH0uCH0B 0HCH0H0QE0H CoHuCHom "O azouo .U.0HIHH H0 00CH0HCH0E 0HCH0H0QE0H CoHuDHow "0 macho .O.0.O|0.v #0 U0CH0HCH0E 0H5u0n0me0p CoHuaHom "d msouw .msou0 H00 0000 00HCB H H00. H HOH.0 000. H ¢5H.0 000. H 00H.0 MHo. H M_Vm..H.0 000. H 500.0 0 HNO. H HOH.0 30. H 500.0 0N0. H H00.0 0N0. H 050.0 000. H 000.0 m 000. H 000.0 5m0. H 000.0 H00. H 500.0 H00. H mmm.0 00H. H 0HH.0 .0 0H O O O N 05000 CoH0H0EQCm mo 00p0CHz 0>HH0HDECO .00udu0u0080u UC0H000H0 H0 00CH0uCH0E CoHusHom CHUHE0uC00 wo.H 0 CH 000.3530 0000F C0onCo 0000Co>HnE0 00 080mg CH00 HC0H03 0>Hu0HHEHHHo C00: .3 0Hn09 137 .HE\CHOHEOUC00 08 mm .HE\CH0HE0HC00 0E 0.NH .HE\CH0HE0uC00 0E 0.m .H0u03 00NHC0H00 .uu0um #0 00>HQE0 onCO\0CHCH0E0H 00>Hnfi0 onCo 0>HHP "0 msouw "O QCoHO um QCOHO "0 macaw O\O O\H O\~ O\O o O\O O\~ O\O o «\O O\H O\~ O\O O\O 0 «\O «\H O\H O\H O\H «\m O\OF 0 ON OH OH RH OH OH OH OH NH HH OH QdOHO >00 CoHu0QCOCH CO MCOHFUHOW .5H C0sounu 0H 0>00 CoHu0QDOCH CHOHEOuC00 CH 0000H0 0000 meoHCO 0000Co>unfi0 CH >uHHOuHoz .HH OHQMB 138 .000\CHO>E0C0x 0E 0.00H .000\CHO>EOC0x 0E 0.0H .000\CHO>E0C0x 0E o.H .000\CH0>E0C0x 0E H.o .000\CH0>E0C0x 0E H0.o .000\H0u03 00NHC0H00 HE H.0 am 05000 "m nacho ”0 05000 “O msouw “0 macho u< msouw .uu0u0 #0 00>HQE0 HUHCO\0CHCH0E0H 00>HQE0 xOHCo 0>HHF O\O O\O O O\O O\O O\O m O\H O\~ O\O OxO O\O O\O O\O O\O O\O O O\O O\O O\O O\O O\O O\O O\O OxO O\O o O\O O\O O\O O\O O\O O\O O\O O\O O\O O\O m O\O O\O O\O O\O O\O O\O O\O O\O O\O O\OP O OH OH RH OH OH OH OH OH HH OH QCOHU >00 CoHu0QCOCH CHHS 00000 HHO 000 CH 00uo0hCH 0000 .5H C050HCH 0H 0>00 COHHOQDOCH Co CHO>E0C0x C0onCo 0000C0>HQE0 CH >uHH0uuoz .NH OHQMB 139 Streptomycin Study Injection of streptomycin beginning on ED 10 produced death of chick embryos receiving 10.0 mg/egg/day on day 11 with no chick embryos surviving past ED 12 (Table 13). Deaths began to occur in chicks receiving streptomycin at a dose of 5.0 mg/egg/day on ED 12, with no chick embryos surviving past ED 14. Deaths began to occur on ED 17 in chick embryos receiving streptomycin at a dose of 1.0 mg/egg/day while little effect was noted in chick embryos receiving a dose between 0.001 to 0.1 mg of streptomycin/egg/day. Ethacrynic Acid Studies Injection of ethacrynic acid (EA) beginning on ED 10 was lethal to all chick embryos receiving a dose of 1.0 mg/egg/day after 1 treatment (Table 14). Two chick embryos receiving EA at a dose of 0.1 mg/egg/day died on ED 14. No chick embryos receiving EA at a dose of 0.001 or 0.01 mg/egg/day died. A second trial was designed to determine a maximal tolerable dose of gentamicin and EA togetheru Combinations of EA at a dose of 0.001 mg/egg/day with gentamicin at a dose between 0.001 to 0.1 mg/egg/day caused death beginning on ED 17 in chick embryos receiving gentamicin at a dose of 0.1 mg/egg/day (Table 15). A few chick embryos receiving combinations of EA at a dose of 0.01 mg/egg/day with gentamicin at doses between 0.001 to 0.1 mg/egg/day died early in the treatment period with no relationship to the dose. Combinations of EA at 0.1 mg/egg/day with gentamicin at 0.001 and 0.01 mg/egg/day resulted in chick embryo mortality during .000\CH0>Eou00Hu0 0E 0.0H "O 050H0 .000\CHO>Eou00Hu0 0E 0.0 "0 0sou0 .000\CHO>Eou00Hu0 0E 0.H "m 0COHO .000\CH0>Eou00H90 0E H.o "0 0:0HO .000\CH0>Eou00Hu0 0E Ho.o "O 050HO .000\CH0>Eou00Hu0 0E Hoo.o "0 0Couw .000\H0u03 00NHC0H00 HE H.0 "4 0souw .pu0um H0 00>HQE0 onCO\0CHCH0E0H 00>H080 onCo 0>HHF 140 O\O O\O O\O O\O 0 O\O O\m O\O O\O O\O O\O O O\O O\O O\O O\O O\O O\O OxO O\O O\O m O\O O\O O\O O\O O\O O\O O\O O\O O\O O O\O O\O O\O O\O OxO O\O O\O O\O O\O o O\O O\O O\O O\O O\O O\O O\O O\O O\O m R\R R\R R\R R\R R\R R\R R\R R\R R\RF 0 OH RH OH OH OH OH OH HH OH 05000 >00 CoHu0nsOCH .5H C000HCU 0H 0>00 CoHu0QDOCH Co CH0>Eou00uu0 CHHB 00000 HH0 000 CH 00000HCH 0000 C0onCo 00HOCo>unfi0 CH >uHH0HHoz .OH 0H00B 141 .000\0H00 0HC>HO0CHO 0E 0.H .000\0H00 0HC>HO0CHO 0E H.0 .000\0H00 0HC>H00CU0 0E Ho.0 .000\0H00 0HC>H00CH0 0E Hoo.o .000\H0u03 000HC0H00 HE H.o .HH000 U0 00>Hnfi0 xOHC0\0CHCH0E0H 00>HQE0 onCO 0>HHF "m 050H0 "0 0:0H0 "0 09000 "0 0sou0 u< 0:0H0 O\O O\O m O\O O\O O\O O\O O\O O\O O\O O\O O\O O O\O O\O O\O O\O O\O O\O O\O O\O O\O 0 O\O O\O O\O O\O O\O O\O O\O O\O O\O m O\R O\R O\R O\R O\R O\R O\O O\O O\OF 0 OH RH OH OH OH OH OH HH OH 0Couo >00 CoHu0000CH CHHB 00000 HH0 000 CH 00u00flCH 0000 C0x0HCO 00H0Co>unfi0 CH >HHHOuHoz .5H C0=oHCu 0H 0>00 CoHu0050CH Co H400 0H00 0HC>H00CH0 .OH 0HQ0B .000\CH0HE0HC00 0E Ho.0 .0H00 0HC>HO0CH0 0E H.o "H 0:000 .000\CH0HE00C00 0E H00.0 .0H00 0HC>HO0CH0 0E H.o "m 090nm .000\CH0HE0uC00 0E H.o .0H00 0HC>HO0000 0E H0.o "0 0sou0 .000\CH0HE0HC00 0E Ho.0 .0H00 0HC>H00Cu0 0E Ho.o "0 0:0H0 .000\CHOHEOHC00 0E H00.0 .0H00 0HC>HO0CH0 0E Ho.0 um 0:0H0 .000\CH0HE0uC00 0E H.0 .0H00 0HC>HO0CH0 0E H00.0 "0 05000 .000\CH0HE0HC00 0C Ho.o .0H00 0HC>H00CH0 0E H00.0 "O 050H0 .000\CH0HE0uC00 0E H00.0 .0H00 0HC>H00CH0 0E H00.0 “m 050H0 .000\H0003 000HC0H00 HE H.o "4 0:0H0 .uu0u0 #0 00>HQE0 onC0\0CHCH0E0H 00>Hnfi0 HOHCO 0>HH_ 142 O\O O\O O\~ O\~ O\~ O\O O\O O\O O\O H O\H O\~ O\O O\O O\O O\O O\O O\O O\O m O\O O\O O\O O\O O\O OxO O\O O\O O\O 0 O\O O\O O\O O\O O\O O\O O\O O\O O\O O O\O O\O O\O O\O O\O OxO O\O O\O O\O O O\O O\O O\O O\O O\O O\O O\O O\O O\O O O\O O\O O\O O\O O\O O\O OxO O\O O\O 0 O\O O\O O\O O\O O\O O\O O\O O\O O\O O O\R O\R O\R O\R O\R O\R O\O O\O O\Or O OH RH OH OH OH OH OH HH OH 0:0H0 >00 C0HH0QDOCH .5H C050HCH 0H 0>00 CoHu0nCOCH C0 CH0HE0HC00 0C0 0H00 0HC>HO0CHO CuHs 00000 HH0 000 CH 00uo0nCH 0000 C0x0HCO 0000C0>H0E0 CH >HHH0pHoz .0H 0HQOB 143 ED 12 to 14 with remaining chick embryos surviving until ED 18 (end of the observation period). DISCUSSION Each of the drugs used in these dose response studies caused death of chick embryos. This allowed easy determination of a maximally tolerated dose to be used for other toxicity studies using the chick embryo. There was no difference between the results of injection.of gentamicin into the air space early in incubation to late in incubation. Thus, when doses do not cause death within one or two days, lethality may be dependent upon a specific stage of development. This could be explained by the onset of enzyme function to bioactivate a protoxicant or it may reflect increasing dependence on an organ system such as the kidneys which may be damaged by the drugs. Also, submersion did not appear to be a method of exposure that provided a consistently predictable dose. Results, even those from the negative control group indicated this method had considerable draw backs. LIST OF REFERENCES LIST OF REFERENCES Abratt R.P., and Blackburn.J.D. (1980) Loss of hearing after misonidazole. 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