Distribution of Labeled in the Inner Ear Lennart Dencker, DVM, Nils Gunnar
Lindquist,
Autoradiography of the inner ear was performed at varying intervals following intravenous injection of either chloroquine tagged with carbon 14 ("C), or an iodine 125 (125l)-labeled analogue of chloroquine, in rats. In pigmented rats a strong accumulation and retention was noted in the melanin-bearing tissues. In the inner ear there was a very high concentration in the melanin-containing tissues, eg, the stria vascularis and the planum semilunatum. A strong retention was found in these structures 13 days after injection. Accumulation was not observed in the endolymph nor in the perilymph. No
accumulation was found in the inner ear of an albino rat. The ototoxic effects of chloroquine may be caused by an accumulation of the drug in the melanin-containing structures in the inner ear, leading to pathological changes in these tissues and secondary lesions in the receptor cells.
for publication May 13, 1974. From the Department of Toxicology, University of Uppsala, Biomedical Center, Uppsala,
Accepted
Sweden.
Reprint requests to the Department of Toxicology, University of Uppsala, Biomedical Center, Box 573, S-751 23 Uppsala, Sweden (Dr. Dencker).
Chloroquine
PhD
high-dose therapy Prolochloroquine nged eye13 damage.
with is known to cause both and inner ear49 The importance of the affinity of chloroquine for melanin as the cause of retinal damage is well estab¬ lished.1 l0 It is possible that the accu¬ mulation of chloroquine on melanin is also the most important factor in its ototoxicity. Melanin is present in the stria vascularis of the cochlea and the planum semilunatum of the am¬ pullae.1115 These structures, as well as the melanin-containing tissues of the eye, form the fluid that nourishes the receptor cells.1617 Damage to these cells may thus occur secondarily to le¬ sions in the melanin-containing cells. The mechanism of chloroquine's ototoxicity may be of more general interest because its ototoxic lesions are similar to those caused by the aminoglycoside group of antibiotics.9 Both hearing and balance difficulties may appear. The symptoms may ap¬ pear weeks or months after drug withdrawal. Although the changes in the receptor cells have attracted the most interest in the literature, lesions in the melanin-containing stria vas-
Downloaded From: http://archotol.jamanetwork.com/ by a Oakland University User on 06/01/2015
cularis have been found almost con¬ stantly in animals treated with these ototoxic drugs.18 In previous whole-body autoradiographic investigations of chloro¬ quine tagged with carbon 14 (14C) in pregnant mice, an accumulation was seen in the inner ear of the pig¬ mented mouse fetuses.2-3 The autoradiographic pattern of the inner ear of adult mice could not be interpreted due to the difficulties in obtaining good sections through the hard tem¬ poral bone. The present investigation compris¬ ing both whole-body and microautoradiography was made in young rats due to their softer temporal bones. In addition to 14C-chloroquine its iodine analogue was studied because of the favorable radiation properties of this analogue in autoradiography. METHODS
14C-chloroquine (ring-3-14C), 4-(4'-diethylamino 1 methylbutylamino) 7 chloroquinoline, specific activity 14.0 microcuries/mg was obtained. The chloro¬ quine base was dissolved in an aqueous so¬ lution of H3P04 with a molar ratio H,P04: chloroquine 2:1, yielding the diphosphate. The
-
=
-
-
-
The (4-(3'-dimethylaminopropylamino)-7iodoquinoline (DAPQ) labeled with iodine 125 (,25I), specific activity 1.0 to 1.6 millicuries/mg was dissolved in 0.33 hydro¬
chloric acid. Twelve young hooded rats of the Lister strain and two young albino rats of the Sprague-Dawley strain were used. The an¬ imals were kept on a complete pellet diet (AB Ewos) at a room temperature of +25 C, with free access to water.
Whole
Body Autoradiography
Six hooded rats, 5 days old, and one al¬ bino rat of the same age were given injec¬ tions in a tail vein of 125I-DAPQ, each animal receiving 10 microcuries, corre¬ sponding to 7.4 to 11.1 gm 4-(3'-dimethyl-
aminopropylamino)-7-iodoquinoline dihydrochloride. The rats were killed by chloroform anesthesia 20 minutes, 1, 4, and 24 hours, and 4 days (one pigmented ani¬ mal plus one albino animal), and 13 days after injection. Two 12-day-old hooded rats each received 20 microcuries of 125I-DAPQ, intravenously, and were killed 24 hours and six days after injection. One 25-dayold hooded rat was given 10 microcuries of 14C-chloroquine injected intravenously, cor¬ responding to 1.15 mg chloroquine diphosphate, and was killed 48 hours after injec¬
tion.
Immediately
after being killed, the ani¬ mounted in a gel of carboxymethyl cellulose, and immersed in nhexane cooled to -78 C with solid carbon dioxide. Sagittal 15µ and 40jum sections through the whole frozen animal were cut on tape (No. 800, Minnesota Mining and Manufacturing Co.) in a microtome at -15 mals
Fig
were
C and dried at the
same
temperature, all
according to Ullberg's whole body autoradiographic technique.19·20 Autoradiograms were made by apposition of the sections to roentgen films (Industrex, Kodak) or nuclear emulsion plates (G 5, IIford). The exposure times varied from three weeks to three months. After expo¬ sure, the sections were separated from the films. The films were processed (Gevaert G 150 developer and Gevaert G 305 fixative). A number of sections were stained in hematoxylin-eosin and mounted (Euparal).
Microautoradiography Two hooded rats, 5 days old, were in¬ jected intravenously with 12T-DAPQ, each animal receiving 40 microcuries. The rats
killed with chloroform anesthesia 48 hours after injection. The temporal bones were rapidly removed and immersed in isopentane cooled with liquid nitrogen. Cryo¬ stat sections, 5µ thick, were cut at —20 C through the inner ear at various levels par¬ allel to the modiolus. The sections were directly picked up on the nuclear emulsion plates (G 5, Ilf ord), according to Appleton 's technique.21 The plates were pretreated by dipping in 10% glycerol in absolute ethanol to increase the stickiness of the emulsion surface, according to Hammarstróm et al.22 The sections and the films were stored in a refrigerated room at —15 C during the ex¬ posure time, which was seven days. After photographic processing and rinsing the sections were stained with hematoxylineosin, passed through a series of increas¬ ing concentrations of ethanol to xylene and finally mounted in Canada balsam. As control, cryostat sections were cut were
1 —Detail of
an autoradiogram (left) with the corresponding section (right) of a pigmented rat 13 days after receiving 10 microcuries of 125I-DAPQ, injected in¬ travenously. There is high accumulation (white areas) in the striae vasculares (hema¬ toxylin-eosin, 13).
young
from the inner ear of a noninjected, 7-dayold hooded rat, and were autoradiographed as described above.
Light Microscopy One hooded rat and one albino rat, both 12 days old, were killed by chloroform anesthesia, and the temporal bones were removed and fixed in 3.5% formaldehyde solution for 24 hours. After paraffin em¬ bedding, 5fim-thick sections were cut through the inner ear at various levels par¬ allel to the modiolus. The sections were mounted on slides, deparaffinized in xylol, and carried through an alcohol series to distilled water, and thereafter the sections were stained either with hematoxylin-eo¬ sin or with the Masson-Fontana method for melanin (reduction of ammoniacal sil¬ ver nitrate to metallic silver).
RESULTS General Observations Twenty minutes after receiving
125I-DAPQ, injected intravenously, most of the labeled substance had al¬
ready left the blood, and was localized in tissues such as the kidney, liver, lung, stomach, salivary glands, spleen, the melanin-containing tis¬ sues of the eye, the meninges, the skin, the hair follicles, and in the in¬ ner ear. Also the adrenal glands, pitu¬ itary gland, and pancreatic islands had initially high levels. From four hours after injection high activity was observed in the thyroid, probably
due to accumulation of free iodine 125. Apart from the high concentra¬ tion in the thyroid, the level in all nonmelanin structures decreased rap¬ idly and from four days the distribu¬ tion picture in the hooded rats was dominated by a strong accumulation and retention in the melanin-bearing structures. No accumulation was found in the corresponding tissues in the albino rat. There was a strong accumulation in the melanin-containing tissues, in¬ cluding the inner ear, 48 hours after 14C-chloroquine was injected intrave¬ nously in a pigmented rat. Inner Ear
After receiving an injection of ei¬ ther 125I-DAPQ or 14C-chloroquine there was a high accumulation at all studied survival times in the inner ear of pigmented rats. In the whole-body autoradiograms,
Downloaded From: http://archotol.jamanetwork.com/ by a Oakland University User on 06/01/2015
there was a very high concentration in the melanin-containing tissues, ie, the stria vascularis (Fig 1 and 2), the modiolus, the planum semilunatum, the walls of the sacculus, the utriculus, and the semicircular ducts. These structures were all possible to recognize in the stained whole-body sections. In the microautoradiograms high uptake was seen in melaninbearing cells, but it was often diffi¬ cult to distinguish the silver grains underneath heavily pigmented cells. No accumulation was observed at any survival time in either the endolymph or the perilymph. No accumulation was found in the inner ear in the al¬ bino rat four days after injection of 125I-DAPQ. Specific accumulation of silver grains over special cells could not be seen in any of the control sections. In sections, cut from the inner ear of a pigmented rat, and stained in hematoxylin-eosin or with the Masson-Fontana method for melanin, pigment-containing cells were found in the structures showing high accu¬ mulation of radioactivity in the autoradiograms (Fig 3). Most of the mela¬ nin-bearing cells in the inner ear were adherent to blood vessels, as well in the epithelia of the stria vas¬ cularis and the planum semilunatum, as in the connective tissues of the modiolus, the walls of the sacculus, the utriculus, and the semicircular ducts. No pigment-containing cells were observed in the inner ear of an albino rat of the same age.
COMMENT The results of this investigation re¬ veal a heavy accumulation and reten¬ tion in the melanin-containing tis¬ sues (eg, the stria vascularis in the cochlea and the planum semilunatum in the ampullae) in the inner ear of the pigmented rats after injection of either 14C-chloroquine or its 125IDAPQ analogue. No accumulation was observed in the endolymph, peri¬ lymph, the sensory cells, or in the nerves. In the albino rat no accumula¬ tion was noted in any structure of the inner ear. The distribution in mice of the io¬ dine 125-analogue was found previ¬ ously to be very similar to that of 14C-
Fig 2.—Microautoradiogram (section + autoradiogram) of the inner ear from a pigmented rat 48 hours after receiving 40 microcuries of 125I-DAPQ, injected intravenously. Very high uptake (black grains) can be seen in several cells in the stria vas¬ cularis (hematoxylin-eosin, 400).
chloroquine.23 This was true also in the present investigation. In our autoradiographic distribu¬ tion studies in pigmented mice,2-3
melanin has been shown to accumu¬ late and retain many substances of a polycyclic nature. This accumulation was not found in the albino animals. A high retention was observed even one year after the administration of a single dose of 14C-chloroquine. It was also noted that not only chloroquine but also all of the aminoglycoside an¬ tibiotics investigated have a strong affinity for melanin in vitro. The ototoxic lesions caused by chloroquine and the aminoglycoside antibiotics have many characteristics
Downloaded From: http://archotol.jamanetwork.com/ by a Oakland University User on 06/01/2015
Fig 3.—Light micrograph
of a Masson-Fontana-stained 5µ section showing mela¬ nin-containing cells in the stria vascularis of a young pigmented rat ( 350).
in common.9 High term therapy are
dosage and longusually involved. a marked tendency
has irreversible. Furthermore, symptoms often show up only after a long latent period—weeks or months after the medication has been discon¬ tinued. These features, especially the latent period, indicate the presence of a strong mechanism of accumulation and retention of the drugs in the inner ear. In the inner ear, melanin is found in the richly vascularized areas, and the melanocytes often surround the blood vessels.1115 Accumulation of ototoxic drugs in the melanin-con¬ taining cells may cause vascular inThe to
damage
be
juries and degenerative changes in the stria vascularis and the planum semilunatum. Both blood vessel dys¬ function and lesions in the epithelial
cells of these tissues may result in a changed composition of the endo¬ lymph. This may secondarily damage the receptor cells. This hypothesis for the mechanism of drug-induced inner ear damage is consistent with that suggested by Hawkins,18 ie, that the primary ototoxic lesion is in the
secretory and reabsorptive tissues, and not in the receptor cells.
Chloroquine is known to simultane¬ ously cause damage to both the eye1"3 and the inner ear.49 The ocular dam¬ age is generally accepted to be caused by the melanin affinity.11" Other ocu¬ lar pigment disturbances, such as con¬ genital retinitis pigmentosa and pig¬ mentary glaucoma, are also known to be combined with deafness.2428 Accumulation and long-term reten-
tion of
chloroquine in the melanincontaining cells in the inner ear may well explain the late onset of the le¬ sions and the relation to high dose/ long-term therapy. This investigation was supported by grants from The Swedish Board for Technical Develop¬ ment and The Swedish Medical Research Council
(B74-14X-2876-05B). The '"I-DAPQ was supplied by Dr. Ray Counsell and Dr. Rodney D. Ice, University of Mich¬ igan, Ann Arbor, Mich.
References 1. Meier-Ruge W: Zur Atiologie und Pathogenese toxischer Arzneimittelnebenwirkungen an der Netzhaut. Klin Monatsbl Augenheilkd 163:155-172, 1973. 2. Lindquist NG, Ullberg S: The melanin affinity of chloroquine and chlorpromazine studied by whole body autoradiography. Acta Pharmacol Toxicol (Kbh) (Suppl 2) 31:1-32, 1972. 3. Lindquist NG: Accumulation of drugs on melanin. Acta Radiol [Diagn] (Suppl) [Stockh] 325:1-92, 1973. 4. Dewar WA, Mann HM: Chloroquine in lupus erythematosus. Lancet 1:780-781, 1954. 5. Scherbel AL, Harrison JW, Atdjian M: Further observations on the use of 4-aminoquinoline compounds in patients with rheumatoid arthritis or related diseases. Cleve Clin Q 25:95-111,1958. 6. Hart CW, Naunton RF: The ototoxicity of chloroquine phosphate. Arch Otolaryngol 80:407\x=req-\ 412, 1964. 7. Toone EC Jr, Hayden GD, Ellman HM:
Ototoxicity
of
chloroquine. Arthritis Rheum
8:475-476, 1965. 8. Sataloff J, Vassallo L: Medical audiology. Arch Otolaryngol 91:208-211, 1970. 9. Sheffield PA, Turner JS Jr: Ototoxic drugs: A review of clinical aspects, histopathologic
changes and
mechanisms of action. South Med J
64:359-363, 1971.
10. Potts AM: The reaction of uveal
pigment
in vitro with polycyclic compounds. Invest Ophthalmol 3:405-416, 1964. 11. Corti A: Recherches sur l'organe de l'oule des mammif\l=e`\res.Z Wiss Zool 3:109-169, 1851. 12. Alexander G: Das Labyrinthpigment des Menschen und der h\l=o"\herenS\l=a"\ugethierenebst Bemerkungen \l=u"\berden feineren Bau des perilymphatischen Gewebes. Arch Mikrosk Anat 58:134-181, 1901. 13. Beck C: Das Pigment der Stria vascularis. Arch Ohren Nasen Kehlkopfheilkd 179:51-58, 1961. 14. Sieber J, Schmidt H: Histologische und histochemische Untersuchungen an h\l=a"\utigenBogeng\l=a"\ngen.Z Laryngol Rhinol Otol 41:46-55, 1962. 15. Savin C: The blood vessels and pigmentary cells of the inner ear. Ann Otol Rhinol Laryngol 74:611-622, 1965. 16. R\l=u"\ediL: Uber die Funktion der Stria vascularis. Pract Otorhinolaryngol (Basel) 13:341\x=req-\ 352, 1951. 17. Rauch S: Entstehung, Transport und Resorption der Innenohrfl\l=u"\ssigkeiten,in Biochemie des H\l=o"\rorgans:Einf\l=u:\hrungin Methoden und Ergebnisse. Stuttgart, Georg Thieme Verlag, 1964. 18. Hawkins JE Jr: Ototoxic mechanisms: A working hypothesis. Audiology 12:383-393, 1973. 19. Ullberg S: Studies on the distribution and
Downloaded From: http://archotol.jamanetwork.com/ by a Oakland University User on 06/01/2015
fate of S35-labelled benzylpenicillin in the body. Acta Radiol [Suppl] (Stockh) 118:1-110, 1954. 20. Ullberg S: Autoradiographic studies on the distribution of labelled drugs in the body, in Proceedings of the Second United Nations International Conference on the Peaceful Uses of Atomic Energy, Geneva. Geneva, United Nations Publication, 1958, vol 24, pp 248-254. 21. Appleton TC: Autoradiography of soluble labelled compounds. J R Microsc Soc 83:277-281, 1964. 22. Hammarstr\l=o"\mL, Appelgren L-E, Ullberg S: Improved method for light microscopy autoradiography with isotopes in water-soluble form. Exp Cell Res 37:608-613, 1965. 23. Counsell RE, Pocha P, Morales JO, et al: Tumor localizing agents: III. Radioiodinated quinoline derivatives. J Pharmacol Sci 56:1042\x=req-\ 1044, 1967. 24. Bietti GB: The result of audiometric examinations in some ocular diseases. Trans Ophthalmol Soc UK 72:343-355, 1952. 25. G\l=a"\rtnerJ: Pigmentglaukom und Innenohrschwerh\l=o"\rigkeit.Ophthalamologica 147:393\x=req-\ 408, 1964. 26. Goode RL, Rafaty FM, Simmons FB: Hearing loss in retinitis pigmentosa. Pediatrics 40:875-880, 1967.
Lindquist,
Autoradiography of the inner ear was performed at varying intervals following intravenous injection of either chloroquine tagged with carbon 14 ("C), or an iodine 125 (125l)-labeled analogue of chloroquine, in rats. In pigmented rats a strong accumulation and retention was noted in the melanin-bearing tissues. In the inner ear there was a very high concentration in the melanin-containing tissues, eg, the stria vascularis and the planum semilunatum. A strong retention was found in these structures 13 days after injection. Accumulation was not observed in the endolymph nor in the perilymph. No
accumulation was found in the inner ear of an albino rat. The ototoxic effects of chloroquine may be caused by an accumulation of the drug in the melanin-containing structures in the inner ear, leading to pathological changes in these tissues and secondary lesions in the receptor cells.
for publication May 13, 1974. From the Department of Toxicology, University of Uppsala, Biomedical Center, Uppsala,
Accepted
Sweden.
Reprint requests to the Department of Toxicology, University of Uppsala, Biomedical Center, Box 573, S-751 23 Uppsala, Sweden (Dr. Dencker).
Chloroquine
PhD
high-dose therapy Prolochloroquine nged eye13 damage.
with is known to cause both and inner ear49 The importance of the affinity of chloroquine for melanin as the cause of retinal damage is well estab¬ lished.1 l0 It is possible that the accu¬ mulation of chloroquine on melanin is also the most important factor in its ototoxicity. Melanin is present in the stria vascularis of the cochlea and the planum semilunatum of the am¬ pullae.1115 These structures, as well as the melanin-containing tissues of the eye, form the fluid that nourishes the receptor cells.1617 Damage to these cells may thus occur secondarily to le¬ sions in the melanin-containing cells. The mechanism of chloroquine's ototoxicity may be of more general interest because its ototoxic lesions are similar to those caused by the aminoglycoside group of antibiotics.9 Both hearing and balance difficulties may appear. The symptoms may ap¬ pear weeks or months after drug withdrawal. Although the changes in the receptor cells have attracted the most interest in the literature, lesions in the melanin-containing stria vas-
Downloaded From: http://archotol.jamanetwork.com/ by a Oakland University User on 06/01/2015
cularis have been found almost con¬ stantly in animals treated with these ototoxic drugs.18 In previous whole-body autoradiographic investigations of chloro¬ quine tagged with carbon 14 (14C) in pregnant mice, an accumulation was seen in the inner ear of the pig¬ mented mouse fetuses.2-3 The autoradiographic pattern of the inner ear of adult mice could not be interpreted due to the difficulties in obtaining good sections through the hard tem¬ poral bone. The present investigation compris¬ ing both whole-body and microautoradiography was made in young rats due to their softer temporal bones. In addition to 14C-chloroquine its iodine analogue was studied because of the favorable radiation properties of this analogue in autoradiography. METHODS
14C-chloroquine (ring-3-14C), 4-(4'-diethylamino 1 methylbutylamino) 7 chloroquinoline, specific activity 14.0 microcuries/mg was obtained. The chloro¬ quine base was dissolved in an aqueous so¬ lution of H3P04 with a molar ratio H,P04: chloroquine 2:1, yielding the diphosphate. The
-
=
-
-
-
The (4-(3'-dimethylaminopropylamino)-7iodoquinoline (DAPQ) labeled with iodine 125 (,25I), specific activity 1.0 to 1.6 millicuries/mg was dissolved in 0.33 hydro¬
chloric acid. Twelve young hooded rats of the Lister strain and two young albino rats of the Sprague-Dawley strain were used. The an¬ imals were kept on a complete pellet diet (AB Ewos) at a room temperature of +25 C, with free access to water.
Whole
Body Autoradiography
Six hooded rats, 5 days old, and one al¬ bino rat of the same age were given injec¬ tions in a tail vein of 125I-DAPQ, each animal receiving 10 microcuries, corre¬ sponding to 7.4 to 11.1 gm 4-(3'-dimethyl-
aminopropylamino)-7-iodoquinoline dihydrochloride. The rats were killed by chloroform anesthesia 20 minutes, 1, 4, and 24 hours, and 4 days (one pigmented ani¬ mal plus one albino animal), and 13 days after injection. Two 12-day-old hooded rats each received 20 microcuries of 125I-DAPQ, intravenously, and were killed 24 hours and six days after injection. One 25-dayold hooded rat was given 10 microcuries of 14C-chloroquine injected intravenously, cor¬ responding to 1.15 mg chloroquine diphosphate, and was killed 48 hours after injec¬
tion.
Immediately
after being killed, the ani¬ mounted in a gel of carboxymethyl cellulose, and immersed in nhexane cooled to -78 C with solid carbon dioxide. Sagittal 15µ and 40jum sections through the whole frozen animal were cut on tape (No. 800, Minnesota Mining and Manufacturing Co.) in a microtome at -15 mals
Fig
were
C and dried at the
same
temperature, all
according to Ullberg's whole body autoradiographic technique.19·20 Autoradiograms were made by apposition of the sections to roentgen films (Industrex, Kodak) or nuclear emulsion plates (G 5, IIford). The exposure times varied from three weeks to three months. After expo¬ sure, the sections were separated from the films. The films were processed (Gevaert G 150 developer and Gevaert G 305 fixative). A number of sections were stained in hematoxylin-eosin and mounted (Euparal).
Microautoradiography Two hooded rats, 5 days old, were in¬ jected intravenously with 12T-DAPQ, each animal receiving 40 microcuries. The rats
killed with chloroform anesthesia 48 hours after injection. The temporal bones were rapidly removed and immersed in isopentane cooled with liquid nitrogen. Cryo¬ stat sections, 5µ thick, were cut at —20 C through the inner ear at various levels par¬ allel to the modiolus. The sections were directly picked up on the nuclear emulsion plates (G 5, Ilf ord), according to Appleton 's technique.21 The plates were pretreated by dipping in 10% glycerol in absolute ethanol to increase the stickiness of the emulsion surface, according to Hammarstróm et al.22 The sections and the films were stored in a refrigerated room at —15 C during the ex¬ posure time, which was seven days. After photographic processing and rinsing the sections were stained with hematoxylineosin, passed through a series of increas¬ ing concentrations of ethanol to xylene and finally mounted in Canada balsam. As control, cryostat sections were cut were
1 —Detail of
an autoradiogram (left) with the corresponding section (right) of a pigmented rat 13 days after receiving 10 microcuries of 125I-DAPQ, injected in¬ travenously. There is high accumulation (white areas) in the striae vasculares (hema¬ toxylin-eosin, 13).
young
from the inner ear of a noninjected, 7-dayold hooded rat, and were autoradiographed as described above.
Light Microscopy One hooded rat and one albino rat, both 12 days old, were killed by chloroform anesthesia, and the temporal bones were removed and fixed in 3.5% formaldehyde solution for 24 hours. After paraffin em¬ bedding, 5fim-thick sections were cut through the inner ear at various levels par¬ allel to the modiolus. The sections were mounted on slides, deparaffinized in xylol, and carried through an alcohol series to distilled water, and thereafter the sections were stained either with hematoxylin-eo¬ sin or with the Masson-Fontana method for melanin (reduction of ammoniacal sil¬ ver nitrate to metallic silver).
RESULTS General Observations Twenty minutes after receiving
125I-DAPQ, injected intravenously, most of the labeled substance had al¬
ready left the blood, and was localized in tissues such as the kidney, liver, lung, stomach, salivary glands, spleen, the melanin-containing tis¬ sues of the eye, the meninges, the skin, the hair follicles, and in the in¬ ner ear. Also the adrenal glands, pitu¬ itary gland, and pancreatic islands had initially high levels. From four hours after injection high activity was observed in the thyroid, probably
due to accumulation of free iodine 125. Apart from the high concentra¬ tion in the thyroid, the level in all nonmelanin structures decreased rap¬ idly and from four days the distribu¬ tion picture in the hooded rats was dominated by a strong accumulation and retention in the melanin-bearing structures. No accumulation was found in the corresponding tissues in the albino rat. There was a strong accumulation in the melanin-containing tissues, in¬ cluding the inner ear, 48 hours after 14C-chloroquine was injected intrave¬ nously in a pigmented rat. Inner Ear
After receiving an injection of ei¬ ther 125I-DAPQ or 14C-chloroquine there was a high accumulation at all studied survival times in the inner ear of pigmented rats. In the whole-body autoradiograms,
Downloaded From: http://archotol.jamanetwork.com/ by a Oakland University User on 06/01/2015
there was a very high concentration in the melanin-containing tissues, ie, the stria vascularis (Fig 1 and 2), the modiolus, the planum semilunatum, the walls of the sacculus, the utriculus, and the semicircular ducts. These structures were all possible to recognize in the stained whole-body sections. In the microautoradiograms high uptake was seen in melaninbearing cells, but it was often diffi¬ cult to distinguish the silver grains underneath heavily pigmented cells. No accumulation was observed at any survival time in either the endolymph or the perilymph. No accumulation was found in the inner ear in the al¬ bino rat four days after injection of 125I-DAPQ. Specific accumulation of silver grains over special cells could not be seen in any of the control sections. In sections, cut from the inner ear of a pigmented rat, and stained in hematoxylin-eosin or with the Masson-Fontana method for melanin, pigment-containing cells were found in the structures showing high accu¬ mulation of radioactivity in the autoradiograms (Fig 3). Most of the mela¬ nin-bearing cells in the inner ear were adherent to blood vessels, as well in the epithelia of the stria vas¬ cularis and the planum semilunatum, as in the connective tissues of the modiolus, the walls of the sacculus, the utriculus, and the semicircular ducts. No pigment-containing cells were observed in the inner ear of an albino rat of the same age.
COMMENT The results of this investigation re¬ veal a heavy accumulation and reten¬ tion in the melanin-containing tis¬ sues (eg, the stria vascularis in the cochlea and the planum semilunatum in the ampullae) in the inner ear of the pigmented rats after injection of either 14C-chloroquine or its 125IDAPQ analogue. No accumulation was observed in the endolymph, peri¬ lymph, the sensory cells, or in the nerves. In the albino rat no accumula¬ tion was noted in any structure of the inner ear. The distribution in mice of the io¬ dine 125-analogue was found previ¬ ously to be very similar to that of 14C-
Fig 2.—Microautoradiogram (section + autoradiogram) of the inner ear from a pigmented rat 48 hours after receiving 40 microcuries of 125I-DAPQ, injected intravenously. Very high uptake (black grains) can be seen in several cells in the stria vas¬ cularis (hematoxylin-eosin, 400).
chloroquine.23 This was true also in the present investigation. In our autoradiographic distribu¬ tion studies in pigmented mice,2-3
melanin has been shown to accumu¬ late and retain many substances of a polycyclic nature. This accumulation was not found in the albino animals. A high retention was observed even one year after the administration of a single dose of 14C-chloroquine. It was also noted that not only chloroquine but also all of the aminoglycoside an¬ tibiotics investigated have a strong affinity for melanin in vitro. The ototoxic lesions caused by chloroquine and the aminoglycoside antibiotics have many characteristics
Downloaded From: http://archotol.jamanetwork.com/ by a Oakland University User on 06/01/2015
Fig 3.—Light micrograph
of a Masson-Fontana-stained 5µ section showing mela¬ nin-containing cells in the stria vascularis of a young pigmented rat ( 350).
in common.9 High term therapy are
dosage and longusually involved. a marked tendency
has irreversible. Furthermore, symptoms often show up only after a long latent period—weeks or months after the medication has been discon¬ tinued. These features, especially the latent period, indicate the presence of a strong mechanism of accumulation and retention of the drugs in the inner ear. In the inner ear, melanin is found in the richly vascularized areas, and the melanocytes often surround the blood vessels.1115 Accumulation of ototoxic drugs in the melanin-con¬ taining cells may cause vascular inThe to
damage
be
juries and degenerative changes in the stria vascularis and the planum semilunatum. Both blood vessel dys¬ function and lesions in the epithelial
cells of these tissues may result in a changed composition of the endo¬ lymph. This may secondarily damage the receptor cells. This hypothesis for the mechanism of drug-induced inner ear damage is consistent with that suggested by Hawkins,18 ie, that the primary ototoxic lesion is in the
secretory and reabsorptive tissues, and not in the receptor cells.
Chloroquine is known to simultane¬ ously cause damage to both the eye1"3 and the inner ear.49 The ocular dam¬ age is generally accepted to be caused by the melanin affinity.11" Other ocu¬ lar pigment disturbances, such as con¬ genital retinitis pigmentosa and pig¬ mentary glaucoma, are also known to be combined with deafness.2428 Accumulation and long-term reten-
tion of
chloroquine in the melanincontaining cells in the inner ear may well explain the late onset of the le¬ sions and the relation to high dose/ long-term therapy. This investigation was supported by grants from The Swedish Board for Technical Develop¬ ment and The Swedish Medical Research Council
(B74-14X-2876-05B). The '"I-DAPQ was supplied by Dr. Ray Counsell and Dr. Rodney D. Ice, University of Mich¬ igan, Ann Arbor, Mich.
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