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Increased susceptibility of male rats to kanamycin-induced cochleotoxicity
Hearing Research 128 (1999) 75^79
Increased susceptibility of male rats to kanamycin-induced cochleotoxicity Charles D. Mills 1 , Benjamin M. Loos 2 , Charles M. Henley * The Bobby R. Alford Department of Otorhinolaryngology and Communicative Sciences, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Received 22 January 1998; received in revised form 8 October 1998; accepted 16 October 1998
Abstract Although clinical observations suggest that males are more susceptible than females to ototoxic drugs, controlled experimental studies investigating gender susceptibility have not been performed. Aminoglycosides initially attack the cochlea's outer hair cells (OHCs). We investigated the effects of the aminoglycoside, kanamycin, on electrophysiological function of OHCs in male and female rats. Animals were grouped by gender and treated with kanamycin (400 mg/kg/day kanamycin base, intramuscular injection) or equivolume normal saline. Administration was continued until distortion product otoacoustic emissions (DPOAEs) suggested a loss in OHC function in kanamycin-treated rats. Males treated with kanamycin showed changes in DPOAE thresholds and amplitudes as early as treatment day 10 which spread to all test frequencies by treatment day 13. In contrast, females treated with kanamycin did not show significant changes in thresholds or amplitudes until treatment day 22. The mechanism of increased male susceptibility to kanamycin cochleotoxicity has not been determined. z 1999 Elsevier Science B.V. All rights reserved. Key words: Gender; Sex; Aminoglycoside; Ototoxicity ; Distortion product otoacoustic emission
1. Introduction The use of aminoglycoside antibiotics as therapeutic agents for the treatment of life-threatening, Gram-negative infections is limited by irreversible ototoxicity (hearing loss and/or vestibular disturbances) and reversible nephrotoxicity. Expression of ototoxicity has been described in several species including man (review by Hawkins, 1976 ; Brown et al., 1985). The sensory hair cells within the inner ear are the primary cellular targets. Within the cochlea, the outer hair cells (OHCs) are * Corresponding author. Present address: Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA. Tel.: +1 (805) 447-7023; Fax: +1 (805) 480-1347; E-mail: [email protected] 1 Present address : Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA. 2 Present address: Department of Otolaryngology, Georgetown University Medical Center, 3800 Reservoir Road N.W., Washington, DC 20007, USA.
the initial, primary sites of the damage caused by these antibiotics (Astbury and Read, 1982). Several factors a¡ect the expression of toxicity including impaired renal function, nutritional status, concomitant treatment with another ototoxic drug, noise exposure and chemical composition (structure) of the individual agents (D'Alonzo and Cantor, 1983; Lautermann et al., 1995). Subject age is another important variable in susceptibility to drug-induced ototoxicity, with very young and very old animals appearing to be more susceptible to cochlear damage from aminoglycosides. Maximum susceptibility corresponds to the period of maturation of the animal's auditory function (Carlier and Pujol, 1980 ; Lenoir et al., 1983; Prieve and Yanz, 1984; Pujol, 1986 ; Henley and Rybak, 1995 ; Henley et al., 1996a). Di¡erences in elimination kinetics, e.g. a longer elimination half-life, in developing vs. mature rats may be responsible for this age-related susceptibility (Henley et al., 1996b). Gender also appears to be important in susceptibility to aminoglycoside toxicity. An association between male sex and de-
0378-5955 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 1 9 0 - 7
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velopment of aminoglycoside ototoxicity in humans has been reported (Barza et al., 1980); however, prior exposure to noisy environments and the fact that patients were receiving several other medications, including diuretics, may have contributed to these observations. In the present study we monitored the physiological e¡ects of the aminoglycoside, kanamycin, on OHC function in age- and strain-matched male and female rats placed in a low level noise environment (ambient vivarium room noise). OHC function was determined using distortion product otoacoustic emissions (DPOAEs) as a sensitive measure of drug-induced hearing loss (Brown et al., 1985; Henley and Rybak, 1995 ; Henley et al., 1996a). DPOAE thresholds and amplitudes were a¡ected adversely at a much earlier time in males than in females. 2. Methods Subjects were adult, pigmented Long-Evans rats (230^310 g; Harlan Sprague Dawley, Indianapolis, IN). Subjects were grouped by gender and treatment as follows : (1) males treated with normal saline, (2) males treated with kanamycin, (3) females treated with normal saline and (4) females treated with kanamycin (n = 5 for each group). All animals had free access to standard lab chow and water and were housed in plastic cages containing corncob bedding. Animals were handled in a humane manner in compliance with The Principles of Laboratory Animal Care (NIH publication No. 80-23, revised 1978). The kanamycin groups received an amount of kanamycin sulfate (Sigma, K-4000), reconstituted in saline,
that was equivalent to 400 mg/kg/day of kanamycin base by an intramuscular injection (i.m.) until DPOAEs suggested a loss in OHC function. The normal saline groups received an equivolume saline (i.m.) for the same duration. The DPOAEs at 2f1 3f2 were elicited using a computer-based digital-signal processor (DSP) operated by customized software provided by Drs. Glen Martin and Brenda Lonsbury-Martin, Miami Ear Institute, Miami, FL. Two equilevel (L1 = L2 ) primary signals (f1 and f2 ) with f2 /f1 = 1.3 were generated. Frequencies examined were : 3, 4, 6, 8, 10, 12, 14 and 16 kHz. The primary tones produced by two separate speakers (Etymotic Research, ER-2) were introduced into the animal's sealed ear canal through an insert earphone speculum where they were acoustically mixed. DPOAE recordings were made with a low-noise microphone (Etymotic Research, ER 10B). The microphone's signal was boosted through a preampli¢er connected to the A/D portion of the DSP device that measured the DPOAEs. Detection threshold and suprathreshold measures in the form of input/output (I/O) functions, were obtained by decreasing the primary tones from 85 to 25 dB SPL, in 5-dB steps. The detection `threshold' was de¢ned as the primarytone level at which the DPOAEs were just distinguishable, at 3 dB above the noise £oor. DPOAEs were measured on treatment days 10, 13, 18, 20 and 22. In order to record DPOAEs, brief anesthesia was induced with an i.m. injection of ketamine:xylazine (40 and 8 mg/kg, respectively). Analysis of variance (ANOVA) with an K level set a priori at 0.05 was used to test for signi¢cant di¡erences in thresholds and amplitudes recorded from male and female rats between the kanamycin and normal saline treatment groups for each frequency.
Fig. 1. DPOAE thresholds (mean þ S.E.M.) as a function of frequency recorded from males treated with normal saline or kanamycin for 13 consecutive days (A), females treated with normal saline or kanamycin for 13 consecutive days (B) and females treated with normal saline or kanamycin for 22 consecutive days (C) (n = 5 for each group). An asterisk indicates signi¢cant di¡erences (P 6 0.05) between animals treated with kanamycin and animals treated with normal saline.
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C.D. Mills et al. / Hearing Research 128 (1999) 75^79
77
Fig. 2. DPOAE I/O functions at 6 kHz illustrating mean amplitudes þ S.E.M. for primary levels ranging from 85 to 25 dB SPL recorded from male (A) and female (B) rats treated with normal saline or kanamycin for 13 days and female rats treated with saline or kanamycin for 22 days (C) (n = 5 for each group). For simplicity, the noise £oor shown represents the mean þ S.E.M. for both groups.
3. Results Male rats began to show an elevation in DPOAE thresholds and a decrease in DPOAE amplitudes at the higher test frequencies (10, 12, 14 and 16 kHz) after 10 consecutive, daily doses of kanamycin. Following 13 consecutive doses, the elevation in thresholds and decrease in amplitudes gradually spread to encompass the lower frequencies as well (panels A in Figs. 1^4). In contrast, female rats treated with kanamycin for 13 days showed no changes in thresholds or amplitudes (panels B in Figs. 1^4). On treatment day 13 males treated with kanamycin showed a statistically signi¢cant elevation in thresholds when compared to males treated with normal saline at all frequencies, except
3 kHz (Fig. 1A). Thresholds shifts ranged from 15 dB at 4 kHz to 30 dB at 6 kHz. Kanamycin-treated female rats showed no signi¢cant elevation in thresholds when compared to females treated with normal saline on treatment day 13 (Fig. 1B). Although kanamycin-treated female rats initially demonstrated a slight increase in thresholds and a decrease in amplitudes at the higher test frequencies by treatment days 18^20, they did not demonstrate a signi¢cant elevation in thresholds when compared to normal saline-treated females until treatment day 22. The magnitude of threshold shifts ranged from 15 dB at 3 kHz to 28 dB at 10 kHz (Fig. 1C). DPOAE amplitudes were signi¢cantly decreased in kanamycin-treated male rats on treatment day 13 (pan-
Fig. 3. DPOAE I/O functions at 8 kHz illustrating mean amplitudes þ S.E.M. for primary levels ranging from 85 to 25 dB SPL recorded from male (A) and female (B) rats treated with normal saline or kanamycin for 13 days and female rats treated with saline or kanamycin for 22 days (C) (n = 5 for each group). For simplicity, the noise £oor shown represents the mean þ S.E.M. for both groups.
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Fig. 4. DPOAE I/O functions at 14 kHz illustrating mean amplitudes þ S.E.M. for primary levels ranging from 85 to 25 dB SPL recorded from male (A) and female (B) rats treated with normal saline or kanamycin for 13 days and female rats treated with saline or kanamycin for 22 days (C) (n = 5 for each group). For simplicity, the noise £oor shown represents the mean þ S.E.M. for both groups.
els A in Figs. 2^4). In contrast, the kanamycin-treated female rats showed no change in amplitudes on the same treatment day (panels B in Figs. 2^4). It was not until treatment day 22 that kanamycin-treated females demonstrated a loss in DPOAE amplitudes (panels C in Figs. 2^4). Although there was no statistical di¡erence in the mean amplitude shift for males treated with kanamycin for 13 days compared to females treated for 22 days (Figs. 2^4), not all of the females demonstrated the same degree of loss in DPOAE amplitudes on treatment day 22. This was evident especially at the lower test frequencies where large variations in amplitudes were observed (Fig. 2C). 4. Discussion OHC function, as determined by DPOAEs, was signi¢cantly a¡ected in male rats treated with kanamycin several days before the same daily dose produced a comparable loss in female rats. Increased thresholds and decreased amplitudes were recorded at the higher test frequencies (10, 12, 14 and 16 kHz) in male rats treated with kanamycin as early as treatment day 10. By treatment day 13, damage had spread such that all test frequencies were equally a¡ected in 100% of male rats (5/5). In contrast, impairment in DPOAEs was not demonstrated in female rats (0/5) treated with the same dose of kanamycin for the same period (13 days). Although continued treatment with kanamycin signi¢cantly a¡ected DPOAEs in females by treatment day 22, the degree of loss was not of the same magnitude in all subjects, i.e., some subjects demonstrated partial preservation in OHC function, especially at the lower test frequencies.
The mechanism of enhanced susceptibility of male rats to kanamycin-induced cochleotoxicity is unknown. Sex-related di¡erences in susceptibility of rats to gentamicin nephrotoxicity have been reported (Bennett et al., 1982). Renal dysfunction was less severe in females than in male rats and cortical concentrations of gentamicin were less in females. Recovery or regeneration was apparent in both sexes; however, functional recovery began earlier in females than males. Although functional di¡erences were noted, histopathological ¢ndings (proximal tubular necrosis followed by epithelial regeneration) were similar in both sexes. Exogenous testosterone administered to adult females did not change the pattern of renal dysfunction nor did castration of prepubertal males. Thus, testosterone did not appear to be responsible for the observed di¡erences. Estrogen effects have not been investigated. While sex-related di¡erences in susceptibility of rats to aminoglycoside nephrotoxicity may be responsible for gender di¡erences in expression of ototoxicity, it is important to note that maximum renal dysfunction was observed after 14 days of aminoglycoside treatment (Bennett et al., 1982) and cochlear dysfunction was apparent in the present study between 10 and 13 days of treatment. However, di¡erent aminoglycosidic agents were used in these studies. The inability of the kidney to excrete kanamycin, leading to higher serum concentrations, may lead to the ear tissues being exposed to higher levels of kanamycin. Therefore, the earlier nephrotoxicity of the males may be partially responsible for their earlier development of ototoxicity. A signi¢cant association between male sex and the development of ototoxicity was reported in a prospective clinical study (Barza et al., 1980). However, it is important to note that many variables may have in£u-
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C.D. Mills et al. / Hearing Research 128 (1999) 75^79
enced these ¢ndings. Many patients had severe underlying diseases and were receiving other drugs concurrently, including other antibiotics, immunosuppressive agents and diuretics. In addition, Barza et al. used combined measures from two forms of toxicity, auditory and vestibular, in order to show a signi¢cant contribution of male gender to toxicity. The present study is the ¢rst to demonstrate a gender di¡erence in susceptibility to aminoglycoside-induced cochleotoxicity. The mechanism of this gender di¡erence in aminoglycoside ototoxicity is undetermined ; however, there are several factors (e.g., serum levels and renal function) which may contribute to the observed ototoxicity. Future studies of sex-dependent differences in aminoglycoside pharmacokinetics should be correlated with the onset and progression of nephrotoxicity (e.g., blood urea nitrogen and creatinine) in order to address the contribution of these important factors to the observed di¡erence in ototoxic susceptibility. Acknowledgments This research was supported by NIDCD Grant DC00656 and The Coker Memorial Research Foundation. We acknowledge Drs. Glen Martin and Brenda Lonsbury-Martin, Miami Ear Institute, Miami, FL, for their assistance and guidance in DPOAE measures. References
79
Barza, M., Lauermann, M.W., Tally, F.P., Gorbach, S.L., 1980. Prospective, randomized trial of netilmicin and amikacin, with emphasis on eighth-nerve toxicity. Antimicrob. Agents Chemother. 17, 707^714. Bennett, W.M., Parker, R.A., Elliott, W.C., Gilbert, D.N., Houghton, D.C., 1982. Sex-related di¡erences in the susceptibility of rats to gentamicin nephrotoxicity. J. Infect. Dis. 145, 370^373. Brown, R.D., Henley, C.M., Penny, J.E., Kupetz, S., 1985. Link between functional and morphological changes in the inner ear: functional changes produced by ototoxic agents and their interactions. Arch. Toxicol. Suppl. 8, 240^250. Carlier, E., Pujol, R., 1980. Supra-normal sensitivity to ototoxic antibiotic of the developing rat cochlea. Arch. Otorhinolaryngol. 226, 129^133. D'Alonzo, B.J., Cantor, A.B., 1983. Ototoxicity: Etiology and Issues. J. Family Pract. 16, 489^494. Hawkins, J.E., 1976. Drug ototoxicity. In: Keidel, W.D., Ne¡, W.D. (Eds.), Handbook of Sensory Physiology, Vol. 5, Springer, Berlin, pp. 707^748. Henley, C.M., Rybak, L.P., 1995. Ototoxicity in developing mammals. Brain Res. Rev. 20, 68^90. Henley, C.M., Weatherly, R.A., Martin, G.K., Lonsbury-Martin, B.L., 1996a. Sensitive developmental periods for kanamycin ototoxic e¡ects on distortion product otoacoustic emissions. Hear. Res. 98, 93^103. Henley, C.M., Weatherly, R.A., Ou, C.-N., Brown, R.D., 1996b. Pharmacokinetics of kanamycin in the developing rat. Hear. Res. 99, 85^90. Lautermann, J., McLaren, J., Schacht, J., 1995. Glutathione protection against gentamicin ototoxicity depends on nutritional status. Hear. Res. 86, 15^24. Lenoir, M., Marot, M., Uziel, A., 1983. Comparative ototoxicity of four aminoglycoside antibiotics during the critical period of cochlear development in the rat. Acta Otolaryngol. Suppl. 405, 1^16. Prieve, B.A., Yanz, J.L., 1984. Age-dependent changes in susceptibility to ototoxic hearing loss. Acta Otolaryngol. 98, 428^438. Pujol, R., 1986. Periods of sensitivity to antibiotic treatment. Acta Otolaryngol. Suppl. 429, 29^33.
Astbury, P.J., Read, N.G., 1982. Kanamycin induced ototoxicity in the laboratory rat. Arch. Toxicol. 50, 267^278.
HEARES 3148 8-2-99
Increased susceptibility of male rats to kanamycin-induced cochleotoxicity Charles D. Mills 1 , Benjamin M. Loos 2 , Charles M. Henley * The Bobby R. Alford Department of Otorhinolaryngology and Communicative Sciences, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Received 22 January 1998; received in revised form 8 October 1998; accepted 16 October 1998
Abstract Although clinical observations suggest that males are more susceptible than females to ototoxic drugs, controlled experimental studies investigating gender susceptibility have not been performed. Aminoglycosides initially attack the cochlea's outer hair cells (OHCs). We investigated the effects of the aminoglycoside, kanamycin, on electrophysiological function of OHCs in male and female rats. Animals were grouped by gender and treated with kanamycin (400 mg/kg/day kanamycin base, intramuscular injection) or equivolume normal saline. Administration was continued until distortion product otoacoustic emissions (DPOAEs) suggested a loss in OHC function in kanamycin-treated rats. Males treated with kanamycin showed changes in DPOAE thresholds and amplitudes as early as treatment day 10 which spread to all test frequencies by treatment day 13. In contrast, females treated with kanamycin did not show significant changes in thresholds or amplitudes until treatment day 22. The mechanism of increased male susceptibility to kanamycin cochleotoxicity has not been determined. z 1999 Elsevier Science B.V. All rights reserved. Key words: Gender; Sex; Aminoglycoside; Ototoxicity ; Distortion product otoacoustic emission
1. Introduction The use of aminoglycoside antibiotics as therapeutic agents for the treatment of life-threatening, Gram-negative infections is limited by irreversible ototoxicity (hearing loss and/or vestibular disturbances) and reversible nephrotoxicity. Expression of ototoxicity has been described in several species including man (review by Hawkins, 1976 ; Brown et al., 1985). The sensory hair cells within the inner ear are the primary cellular targets. Within the cochlea, the outer hair cells (OHCs) are * Corresponding author. Present address: Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA. Tel.: +1 (805) 447-7023; Fax: +1 (805) 480-1347; E-mail: [email protected] 1 Present address : Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA. 2 Present address: Department of Otolaryngology, Georgetown University Medical Center, 3800 Reservoir Road N.W., Washington, DC 20007, USA.
the initial, primary sites of the damage caused by these antibiotics (Astbury and Read, 1982). Several factors a¡ect the expression of toxicity including impaired renal function, nutritional status, concomitant treatment with another ototoxic drug, noise exposure and chemical composition (structure) of the individual agents (D'Alonzo and Cantor, 1983; Lautermann et al., 1995). Subject age is another important variable in susceptibility to drug-induced ototoxicity, with very young and very old animals appearing to be more susceptible to cochlear damage from aminoglycosides. Maximum susceptibility corresponds to the period of maturation of the animal's auditory function (Carlier and Pujol, 1980 ; Lenoir et al., 1983; Prieve and Yanz, 1984; Pujol, 1986 ; Henley and Rybak, 1995 ; Henley et al., 1996a). Di¡erences in elimination kinetics, e.g. a longer elimination half-life, in developing vs. mature rats may be responsible for this age-related susceptibility (Henley et al., 1996b). Gender also appears to be important in susceptibility to aminoglycoside toxicity. An association between male sex and de-
0378-5955 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 1 9 0 - 7
HEARES 3148 8-2-99
76
C.D. Mills et al. / Hearing Research 128 (1999) 75^79
velopment of aminoglycoside ototoxicity in humans has been reported (Barza et al., 1980); however, prior exposure to noisy environments and the fact that patients were receiving several other medications, including diuretics, may have contributed to these observations. In the present study we monitored the physiological e¡ects of the aminoglycoside, kanamycin, on OHC function in age- and strain-matched male and female rats placed in a low level noise environment (ambient vivarium room noise). OHC function was determined using distortion product otoacoustic emissions (DPOAEs) as a sensitive measure of drug-induced hearing loss (Brown et al., 1985; Henley and Rybak, 1995 ; Henley et al., 1996a). DPOAE thresholds and amplitudes were a¡ected adversely at a much earlier time in males than in females. 2. Methods Subjects were adult, pigmented Long-Evans rats (230^310 g; Harlan Sprague Dawley, Indianapolis, IN). Subjects were grouped by gender and treatment as follows : (1) males treated with normal saline, (2) males treated with kanamycin, (3) females treated with normal saline and (4) females treated with kanamycin (n = 5 for each group). All animals had free access to standard lab chow and water and were housed in plastic cages containing corncob bedding. Animals were handled in a humane manner in compliance with The Principles of Laboratory Animal Care (NIH publication No. 80-23, revised 1978). The kanamycin groups received an amount of kanamycin sulfate (Sigma, K-4000), reconstituted in saline,
that was equivalent to 400 mg/kg/day of kanamycin base by an intramuscular injection (i.m.) until DPOAEs suggested a loss in OHC function. The normal saline groups received an equivolume saline (i.m.) for the same duration. The DPOAEs at 2f1 3f2 were elicited using a computer-based digital-signal processor (DSP) operated by customized software provided by Drs. Glen Martin and Brenda Lonsbury-Martin, Miami Ear Institute, Miami, FL. Two equilevel (L1 = L2 ) primary signals (f1 and f2 ) with f2 /f1 = 1.3 were generated. Frequencies examined were : 3, 4, 6, 8, 10, 12, 14 and 16 kHz. The primary tones produced by two separate speakers (Etymotic Research, ER-2) were introduced into the animal's sealed ear canal through an insert earphone speculum where they were acoustically mixed. DPOAE recordings were made with a low-noise microphone (Etymotic Research, ER 10B). The microphone's signal was boosted through a preampli¢er connected to the A/D portion of the DSP device that measured the DPOAEs. Detection threshold and suprathreshold measures in the form of input/output (I/O) functions, were obtained by decreasing the primary tones from 85 to 25 dB SPL, in 5-dB steps. The detection `threshold' was de¢ned as the primarytone level at which the DPOAEs were just distinguishable, at 3 dB above the noise £oor. DPOAEs were measured on treatment days 10, 13, 18, 20 and 22. In order to record DPOAEs, brief anesthesia was induced with an i.m. injection of ketamine:xylazine (40 and 8 mg/kg, respectively). Analysis of variance (ANOVA) with an K level set a priori at 0.05 was used to test for signi¢cant di¡erences in thresholds and amplitudes recorded from male and female rats between the kanamycin and normal saline treatment groups for each frequency.
Fig. 1. DPOAE thresholds (mean þ S.E.M.) as a function of frequency recorded from males treated with normal saline or kanamycin for 13 consecutive days (A), females treated with normal saline or kanamycin for 13 consecutive days (B) and females treated with normal saline or kanamycin for 22 consecutive days (C) (n = 5 for each group). An asterisk indicates signi¢cant di¡erences (P 6 0.05) between animals treated with kanamycin and animals treated with normal saline.
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77
Fig. 2. DPOAE I/O functions at 6 kHz illustrating mean amplitudes þ S.E.M. for primary levels ranging from 85 to 25 dB SPL recorded from male (A) and female (B) rats treated with normal saline or kanamycin for 13 days and female rats treated with saline or kanamycin for 22 days (C) (n = 5 for each group). For simplicity, the noise £oor shown represents the mean þ S.E.M. for both groups.
3. Results Male rats began to show an elevation in DPOAE thresholds and a decrease in DPOAE amplitudes at the higher test frequencies (10, 12, 14 and 16 kHz) after 10 consecutive, daily doses of kanamycin. Following 13 consecutive doses, the elevation in thresholds and decrease in amplitudes gradually spread to encompass the lower frequencies as well (panels A in Figs. 1^4). In contrast, female rats treated with kanamycin for 13 days showed no changes in thresholds or amplitudes (panels B in Figs. 1^4). On treatment day 13 males treated with kanamycin showed a statistically signi¢cant elevation in thresholds when compared to males treated with normal saline at all frequencies, except
3 kHz (Fig. 1A). Thresholds shifts ranged from 15 dB at 4 kHz to 30 dB at 6 kHz. Kanamycin-treated female rats showed no signi¢cant elevation in thresholds when compared to females treated with normal saline on treatment day 13 (Fig. 1B). Although kanamycin-treated female rats initially demonstrated a slight increase in thresholds and a decrease in amplitudes at the higher test frequencies by treatment days 18^20, they did not demonstrate a signi¢cant elevation in thresholds when compared to normal saline-treated females until treatment day 22. The magnitude of threshold shifts ranged from 15 dB at 3 kHz to 28 dB at 10 kHz (Fig. 1C). DPOAE amplitudes were signi¢cantly decreased in kanamycin-treated male rats on treatment day 13 (pan-
Fig. 3. DPOAE I/O functions at 8 kHz illustrating mean amplitudes þ S.E.M. for primary levels ranging from 85 to 25 dB SPL recorded from male (A) and female (B) rats treated with normal saline or kanamycin for 13 days and female rats treated with saline or kanamycin for 22 days (C) (n = 5 for each group). For simplicity, the noise £oor shown represents the mean þ S.E.M. for both groups.
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Fig. 4. DPOAE I/O functions at 14 kHz illustrating mean amplitudes þ S.E.M. for primary levels ranging from 85 to 25 dB SPL recorded from male (A) and female (B) rats treated with normal saline or kanamycin for 13 days and female rats treated with saline or kanamycin for 22 days (C) (n = 5 for each group). For simplicity, the noise £oor shown represents the mean þ S.E.M. for both groups.
els A in Figs. 2^4). In contrast, the kanamycin-treated female rats showed no change in amplitudes on the same treatment day (panels B in Figs. 2^4). It was not until treatment day 22 that kanamycin-treated females demonstrated a loss in DPOAE amplitudes (panels C in Figs. 2^4). Although there was no statistical di¡erence in the mean amplitude shift for males treated with kanamycin for 13 days compared to females treated for 22 days (Figs. 2^4), not all of the females demonstrated the same degree of loss in DPOAE amplitudes on treatment day 22. This was evident especially at the lower test frequencies where large variations in amplitudes were observed (Fig. 2C). 4. Discussion OHC function, as determined by DPOAEs, was signi¢cantly a¡ected in male rats treated with kanamycin several days before the same daily dose produced a comparable loss in female rats. Increased thresholds and decreased amplitudes were recorded at the higher test frequencies (10, 12, 14 and 16 kHz) in male rats treated with kanamycin as early as treatment day 10. By treatment day 13, damage had spread such that all test frequencies were equally a¡ected in 100% of male rats (5/5). In contrast, impairment in DPOAEs was not demonstrated in female rats (0/5) treated with the same dose of kanamycin for the same period (13 days). Although continued treatment with kanamycin signi¢cantly a¡ected DPOAEs in females by treatment day 22, the degree of loss was not of the same magnitude in all subjects, i.e., some subjects demonstrated partial preservation in OHC function, especially at the lower test frequencies.
The mechanism of enhanced susceptibility of male rats to kanamycin-induced cochleotoxicity is unknown. Sex-related di¡erences in susceptibility of rats to gentamicin nephrotoxicity have been reported (Bennett et al., 1982). Renal dysfunction was less severe in females than in male rats and cortical concentrations of gentamicin were less in females. Recovery or regeneration was apparent in both sexes; however, functional recovery began earlier in females than males. Although functional di¡erences were noted, histopathological ¢ndings (proximal tubular necrosis followed by epithelial regeneration) were similar in both sexes. Exogenous testosterone administered to adult females did not change the pattern of renal dysfunction nor did castration of prepubertal males. Thus, testosterone did not appear to be responsible for the observed di¡erences. Estrogen effects have not been investigated. While sex-related di¡erences in susceptibility of rats to aminoglycoside nephrotoxicity may be responsible for gender di¡erences in expression of ototoxicity, it is important to note that maximum renal dysfunction was observed after 14 days of aminoglycoside treatment (Bennett et al., 1982) and cochlear dysfunction was apparent in the present study between 10 and 13 days of treatment. However, di¡erent aminoglycosidic agents were used in these studies. The inability of the kidney to excrete kanamycin, leading to higher serum concentrations, may lead to the ear tissues being exposed to higher levels of kanamycin. Therefore, the earlier nephrotoxicity of the males may be partially responsible for their earlier development of ototoxicity. A signi¢cant association between male sex and the development of ototoxicity was reported in a prospective clinical study (Barza et al., 1980). However, it is important to note that many variables may have in£u-
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C.D. Mills et al. / Hearing Research 128 (1999) 75^79
enced these ¢ndings. Many patients had severe underlying diseases and were receiving other drugs concurrently, including other antibiotics, immunosuppressive agents and diuretics. In addition, Barza et al. used combined measures from two forms of toxicity, auditory and vestibular, in order to show a signi¢cant contribution of male gender to toxicity. The present study is the ¢rst to demonstrate a gender di¡erence in susceptibility to aminoglycoside-induced cochleotoxicity. The mechanism of this gender di¡erence in aminoglycoside ototoxicity is undetermined ; however, there are several factors (e.g., serum levels and renal function) which may contribute to the observed ototoxicity. Future studies of sex-dependent differences in aminoglycoside pharmacokinetics should be correlated with the onset and progression of nephrotoxicity (e.g., blood urea nitrogen and creatinine) in order to address the contribution of these important factors to the observed di¡erence in ototoxic susceptibility. Acknowledgments This research was supported by NIDCD Grant DC00656 and The Coker Memorial Research Foundation. We acknowledge Drs. Glen Martin and Brenda Lonsbury-Martin, Miami Ear Institute, Miami, FL, for their assistance and guidance in DPOAE measures. References
79
Barza, M., Lauermann, M.W., Tally, F.P., Gorbach, S.L., 1980. Prospective, randomized trial of netilmicin and amikacin, with emphasis on eighth-nerve toxicity. Antimicrob. Agents Chemother. 17, 707^714. Bennett, W.M., Parker, R.A., Elliott, W.C., Gilbert, D.N., Houghton, D.C., 1982. Sex-related di¡erences in the susceptibility of rats to gentamicin nephrotoxicity. J. Infect. Dis. 145, 370^373. Brown, R.D., Henley, C.M., Penny, J.E., Kupetz, S., 1985. Link between functional and morphological changes in the inner ear: functional changes produced by ototoxic agents and their interactions. Arch. Toxicol. Suppl. 8, 240^250. Carlier, E., Pujol, R., 1980. Supra-normal sensitivity to ototoxic antibiotic of the developing rat cochlea. Arch. Otorhinolaryngol. 226, 129^133. D'Alonzo, B.J., Cantor, A.B., 1983. Ototoxicity: Etiology and Issues. J. Family Pract. 16, 489^494. Hawkins, J.E., 1976. Drug ototoxicity. In: Keidel, W.D., Ne¡, W.D. (Eds.), Handbook of Sensory Physiology, Vol. 5, Springer, Berlin, pp. 707^748. Henley, C.M., Rybak, L.P., 1995. Ototoxicity in developing mammals. Brain Res. Rev. 20, 68^90. Henley, C.M., Weatherly, R.A., Martin, G.K., Lonsbury-Martin, B.L., 1996a. Sensitive developmental periods for kanamycin ototoxic e¡ects on distortion product otoacoustic emissions. Hear. Res. 98, 93^103. Henley, C.M., Weatherly, R.A., Ou, C.-N., Brown, R.D., 1996b. Pharmacokinetics of kanamycin in the developing rat. Hear. Res. 99, 85^90. Lautermann, J., McLaren, J., Schacht, J., 1995. Glutathione protection against gentamicin ototoxicity depends on nutritional status. Hear. Res. 86, 15^24. Lenoir, M., Marot, M., Uziel, A., 1983. Comparative ototoxicity of four aminoglycoside antibiotics during the critical period of cochlear development in the rat. Acta Otolaryngol. Suppl. 405, 1^16. Prieve, B.A., Yanz, J.L., 1984. Age-dependent changes in susceptibility to ototoxic hearing loss. Acta Otolaryngol. 98, 428^438. Pujol, R., 1986. Periods of sensitivity to antibiotic treatment. Acta Otolaryngol. Suppl. 429, 29^33.
Astbury, P.J., Read, N.G., 1982. Kanamycin induced ototoxicity in the laboratory rat. Arch. Toxicol. 50, 267^278.
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