- Email: [email protected]
Transplantin blocks voltage-dependent calcium current in isolated cochlear outer hair cells but is not ototoxic in vivo
BRAIN RESEARCH ELSEVIER
Brain Research 697 (1995) 276-279
Short communication
Transplatin blocks voltage-dependent calcium current in isolated cochlear outer hair cells but is not ototoxic in vivo Takehisa Saito *, Takehito Yamamoto, Zhi Jian Zhang, Takechiyo Yamada, Yasuhiro Manabe, Hitoshi Saito Department of Otolaryngology, Fukui Medical School, Matsuoka-cho, Yoshida-gun, 910-11 Fukui, Japan Accepted 25 July 1995
Abstract
The effects of cisplatin (cis-DDP) and transplatin (trans-DDP) on the voltage-dependent calcium current (/ca) of outer hair cells isolated from the guinea pig cochlea were compared using the whole-cell patch-clamp technique. Both cis-DDP and trans-DDP blocked Ica in a concentration-dependent manner. Trans-DDP did not show ototoxicity or nephrotoxicity in vivo. From these results, it was suggested that the effect of cis-DDP on Ica does not directly lead to cell death. Keywords: Transplatin; Cisplatin; Ototoxicity; Cochlear outer hair cell; Whole-cell patch-clamp; Voltage-dependent calcium channel; Compound action potential
The precise ototoxic mechanism of cisplatin has not yet been demonstrated. As one of the acute ototoxic mechanisms, blocking of the calcium channel [11,16] or mechano-electric transduction channel [4] of the inner ear sensory hair cells in vitro has been reported. These blocking actions on the calcium channel may affect the first step of sound perception system in the cochlea and produce functional disorders [5]. There are many drugs, however, which can affect the voltage-dependent calcium channel, such as calcium channel blockers and heavy metals [8], and some of these do not induce ototoxicity in vivo. Although cisplatin has potent ototoxicity in vivo [9,12], cisplatin did not reduce outer hair cell (OHC) viability in vitro within 6 h after incubation as previously reported [11]. It was previously reported that trans-isomer transplatin has no anti-tumor sensitivity or nephrotoxicity [13]. In the present study, blocking action on the voltage-dependent calcium channel in vitro and the ototoxicity and nephrotoxicity in vivo were compared in cisplatin and transplatin. The toxicities of cis- and transplatin in vivo and in vitro were compared to verify whether these blocking actions at the cell membrane level could lead to chronic toxicity or cell death. Albino guinea pigs (250-400 g) exibiting normal Preyer
* Corresponding author. Fax: (81) (776) 61-8118. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 9 9 8 - 1
reflex were used. The auditory bullae were removed after decapitation and strips of the organ of Corti were collected. The preparation of OHCs was treated enzymatically in Krebs solution containing 500 protease U / m l of dispase (Godo Shusei) for 30-60 s at room temperature. The OHCs in the second and third turns were dissociated mechanically by gentle pipetting. Whole-cell recordings with patch-clamp pipettes were performed as previously described [2]. The external solution contained choline-C1 140 mM, CsC1 5 mM, CaC12 10 mM, HEPES 10 mM and glucose 140 mM. The internal solution contained N-methyl-o-glucamine(NMG)-C1 30 mM, NMG-aspartate 90 mM, TEA-C1 30 mM, CaC12 0.5 mM, MgC12 5 mM, EGTA 5 mM, ATP (disodium salt) 5 mM, cAMP 0.2 mM, GTP (sodium salt) 1 mM and HEPES 10 mM. The pH of external and internal solutions was adjusted to 7.4 and 7.2 with Tris-OH respectively. The voltage-dependent K + currents were blocked by a pipette solution containing TEA + and NMG + instead of K +. Leakage current was subtracted by multiplying the current evoked by - 10 mV voltage command. Cisplatin (cis-DDP or cis-platinum (II) diammine dichloride, Sigma) and transplatin (trans-DDP or trans-platinum (II) diammine dichloride, Sigma) were dissolved in an external solution just before use. Drugs were applied by the use of Y-tube method, by which the solution surrounding a dissociated cell could be completely exchanged within 20 ms [6].
T. Saito et al. / Brain Research 697 (1995) 276-279
Calcium current amplitude was measured before and after the application of cis-DDP or trans-DDP. Twenty-two animals were divided into 3 groups and administered cis-DDP or trans-DDP as follows. Group 1 consisted of 8 guinea pigs treated intramuscularly with 7.5 m g / k g of cis-DDP at an interval of 5 days (total 15 m g / k g ) , group 2 consisted of 9 animals treated with 30 m g / k g of transplatin (total 60 m g / k g ) , and group 3 (control group) consisted of 5 animals treated with 7.5 m l / k g of saline. From the preliminary experiment, 7.5 m g / k g of trans-DDP had not caused functional or morphological damage to the cochlea or kidney (data not shown). Two days after the final administration, guinea pigs were anesthetized with pentobarbital. They underwent tracheostomy and were ventilated with a respirator. The right cochlea was then exposed by a submandibular approach and a needle electrode was placed on the bony surface of the cochlea near the round window. N 1 thresholds of compound action potential (CAP) were measured using filtered clicks at 4 and 8 kHz. After functional observation, cochleae were fixed by 1% osmium tetroxide (0.1 M phosphate buffer, pH7.4) for 1 h. After the surface preparation, 50 OHCs in each row in the middle part of each turn of the cochlea were counted and cochleograms were made. In other groups (control group (n = 5), cis-DDP group (n = 5) and trans-DDP group (n = 8)), serum was taken from the heart and blood urea nitrogen (BUN) and creatinine levels were measured to assess renal function. Analysis of the significance of differences was calculated by Student's t-test. The inhibitory actions of cis-DDP and trans-DDP progressed time-dependently and it took 3 to 4 min to reach a steady state. After washing out the drugs, the current amplitude gradually recovered toward the control level. Holding potential (V H) was - 7 0 mV. Fig. 1 shows the current-voltage relationships of the Ca 2÷ current (/ca) in the presence and absence of 10 -3 M trans-DDP. Current
MP(m~O
.
o
so
VN
I~
lcontrol r t t m ' v v p 10"3M -100
I(pA) Fig. 1. Effect of trans-DDP on the Ca 2+ current (/Ca)' Holding potential (V H) was - 7 0 mV. I - V relationships of /Ca in the presence or absence of 10 -3 M trans-DDP indicated the inhibitory effect of trans-DDP on /Ca in cochlear outer hair cells of the guinea pig.
277
1.0 0.9
g .-i t~
0.8
(o oisov
o
i
0.7
LOIra~-DDP~
0.6 0.5
I
10 -s
|
i
,
m
. . . .
i
.
.
.
.
.
.
.
.
10 -4
i
10 -3
Concentration of antagonists (M) Fig. 2. Concentration-inhibition curves of antagonists for/ca. Each point is the average of 4 to 7 cells and vertical bars represent + S.E.M.
amplitude was measured after the application of the drug for 4 min, at which time a steady state of inhibition had been established. It has already been reported that cis-DDP blocked the voltage-dependent Ca 2÷ channel [16] and this Ca z+ channel in OHCs of guinea pig was of the L-type [7]. In the present study, trans-DDP also blocked voltage-dependent Ca 2÷ channel and reversibly suppressed the/ca to almost the same degree as cis-DDP. That is to say that cisand trans-DDP acted as Ca 2÷ channel antagonists and reduced the amplitudes of the /ca" Fig. 2 shows the concentration-inhibition curves of cis- and trans-DDP on the /Ca. Both of these drugs suppressed the /Ca in a concentration-dependent manner. The threshold concentration of these drugs depressing the /Ca was 10 -4 M. The N 1 thresholds of CAP in animals treated with cis-DDP was significantly elevated to 80 dB (4 kHz: 58 _+ 16.1 dB ( P < 0.001), 8 kHz: 67.6 _ 20.6 dB ( P < 0.001)). However, animals treated with trans-DDP showed almost the same thresholds (4 kHz: 12 _+ 3.0 dB, 8 kHz: 20 _+ 3.2 dB) as the control group (4 kHz: 13 _+ 3.8 dB, 8 kHz: 23 +_ 3.6 dB) (Fig. 3), suggesting that trans-DDP did not affect hearing in the guinea pig. Fig. 4 shows the cochleograms of the animals exposed to cis-DDP and trans-DDP. In the cis-DDP group, the viabilities of OHCs were significantly decreased in all four tums of the cochlea compared with those in the trans-DDP group. Viability in the basal turn was 63 _+ 18.5%. These morphological alterations appeared to concur with the electrophysiological data. BUN and creatinine levels ( m g / d l ) in serum were significantly elevated in the cis-DDP group (BUN; 115.1 _+ 9.2, Creatinine; 1.9 _+ 0.7) compared with those in the control group (BUN; 19.9 _+ 1.2, Creatinine; 0.3 +_ 0.1). In the trans-DDP group, BUN level (19.9 _+ 1.2) showed no significant difference compared with the control group, whereas the creatinine level was slightly increased (0.4 _ 0.1 ( P < 0.05)). From the present study, it has been clarified that transDDP also blocked voltage-dependent /Ca to almost the same degree as cis-DDP. This blocking action has also
T. Saito et al. / Brain Research 697 (1995) 276-279
278
been induced by aminoglycoside antibiotics such as neomycin, gentamicin, kanamycin, and streptomycin at a high concentration of 1 mM [8] as same concentration as trans-DDP observed in this study. Furthermore, Ca 2+ channel blockers and heavy metals blocked /Ca [8]. These Ca 2÷ channel antagonists block the influx of Ca 2+ through the L-type Ca 2÷ channel in OHCs, resulting in a reduction of [Ca 2+ ]i elevation. Consequently, the Ca2+-activated K ÷ channel was suppressed and the amplitude of microphonic potential decreased concomitantly with a depolarization of the membrane potential [8]. However, in vivo experiment is necessary to support this theory. Because these observations were performed in vitro using isolated outer hair cells of the guinea pig cochlea, the environment around the cells and the condition of the cell after isolation are extremely different from those in vivo. Concerning in vivo experiment using the perilymphatic perfusion method, Ca 2+ channel blocker, nimodipine, has reduced the cochlear microphonic potential (CM) and negative summating potential ( - S P ) [1]. In addition, perilymphatic perfusion of gentamicin (GM) lowered the magnitude of CM [15] and cis-DDP caused a large loss of N I threshold sensitivity of auditory brainstem responses (ABRs) [5]. McAlpine and Jonestone [5] suggested that the basolateral walls of the hair cells, the synapses and the neural elements themselves, which were in close proximity to the perilymph of the scala tympani, were not directly affected by cis-DDP. Because a high concentration of cis-DDP or GM (3 mM) was required in scala tympani perfusion to cause any significant hearing losses, whereas scala media iontophoresis of 5 /zM cis-DDP caused a immediate loss of sensitivity in the N 1 response. Therefore, they considered that N 1 threshold elevation was caused
dB SPL 80-:
41fflz
&
60. 402o-
v
J~
o SPL. 80.
8Xnz
l
60,
4o$
2o-
•
i: ciJ-I~t~ nwm-Dll~
Fig. 3. The N 1 thresholds of C A P (4 and 8 k H z filtered clicks) after treatments with c i s - D D P and trans-DDP. The thresholds in the c i s - D D P g r o u p w e r e significantly elevated c o m p a r e d with those in the control and t r a n s - D D P groups.
(%) 10~
©
6(3
'~ •~
413
[ ] Cis-DDP • Trans-DDP • p < 0.05 ** p < 0.0l
20 0 T1
T2
T3
T4
Fig. 4. The c o c h l e o g r a m s after treatments with c i s - D D P and trans-DDP. In the c i s - D D P group, the viabilities of O H C s were significantly decreased in all four turns c o m p a r e d with those in the t r a n s - D D P group. T1 = Basal turn; T2 = second turn; T3 = third turn; T 4 - apical turn. Vertical bars represent + S.D.
by blocking the mechano-electrical transduction (MET) channel on the apical surface of the OHCs. In this context, Kimitsuki et al. [4] reported that cis-DDP blocked MET channel in isolated chick cochlear hair cell at a concentration of 1 mM using a whole-cell patch-clamp technique. This concentration was higher by three orders of magnitude than that concluded under the experiment by scala media iontophoresis. For this reason, it has been explained that the potential across the apical membrane of the hair cell in vivo was much higher than that in vitro because of the existence of the endocochlear potential, and higher concentration of cis-DDP might be necessary for the drug to act on the MET channel in isolated hair cells. Thus, it is very difficult to extrapolate in vitro and in vivo observations. The perilymphatic concentration of platinum was 5 /xM even after a single intramuscular administration with large dose of 10 m g / k g cis-DDP [10]. This value was lower by three orders of magnitude than the concentration necessary to reduce the CM by scala tympani perfusion in vivo or to block the voltage-dependent Ca 2÷ channel and MET channel in vitro. These data indicate that a very high dose of cis-DDP by general administration is necessary to affect these channels in vivo. If platinum compounds exert their toxic effect solely as heavy metals, trans-DDP should be similar in toxicity to cis-DDP. This hypothesis was not supported by our functional and morphological studies. Karen et al. [3] have reported that platinum values in organs such as the kidney, liver, and small intestine after trans-DDP treatment were much higher than those after treatment with cis-DDP. The perilymphatic concentrations of platinum after general administration of equal doses of cis-DDP and trans-DDP were proven to be almost the same by our preliminary study (data not shown). However, trans-DDP did not have ototoxicity in vivo even after the administration of a high dose (30 m g / k g ) . Trans-DDP, administered at total doses two- to four-fold (34 ~ 56 m g / k g ) those of cis-DDP, was also non-nephrotoxic on histological and functional assess-
T. Saito et al. / B r a i n Research 697 (1995) 276-279
ment [3]. Regarding nephrotoxicity, it has been suggested that some form of sterical interaction of cis-DDP with cellular constituents, which differed from that of transDDP, was essential for toxicity [3]. Candidates for this interaction might include DNA, RNA or proteins. Recent studies suggested that mitochondrial DNA in the renal cell might be affected by cis-DDP [14], although it has not yet been clarified whether this effect applied to cochlear hair cells. In conclusion, trans-DDP blocked voltage-dependent /Ca to almost the same degree as cis-DDP using a whole-cell patch-clamp method. However, trans-DDP did not show ototoxicity or nephrotoxicity in vivo, suggesting that the effect on the voltage-dependent Ca 2+ channel by cis-DDP does not directly cause cell death. Further investigation will be needed to provide more insight into the mechanisms of ototoxicity by cis-DDP.
References
[5] [6]
[7]
[8]
[9]
[10]
[11]
[12] [13]
[1] Bobbin, R.P., Jadtreboff, P.J., Fallon, M. and Littman, T., Nimodipine, an L-channel antagonist, reverses the negative summating potential recorded from the guinea pig cochlea, Hear. Res., 46 (1990) 277-288. [2] Kakehata, S., Nakagawa, T., Takasaka T. and Akaike, N., Cellular mechanism of acetylcholine-induced response in dissociated outer hair cells of guinea-pig cochlea, J. Physiol., 463 (1993) 227-244. [3] Karen, S.B., Harrington D.A., Long, D.A. and Jackson J.E., Relative lack of toxicity of transplatin compared with cisplatin in rodants, J. Comp. Pathol., 105 (1991) 367-375. [4] Kimitsuki, T., Nakagawa, T., Hisashi, K., Komune, S. and
[14] [15]
[16]
279
Komiyama S., Cisplatin blocks mechano-electric transducer current in chick cochlear hair cells, Hear. Res., 71 (1993) 64-68. McAlpine, D. and Johnstone B.M., The ototoxic mechanism of cisplatin, Hear. Res., 47 (1991) 191-204. Murase, K., Ryu, P.D. and Randic, M., Excitatory and inhibitory amino acids- and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons, Neurosci. Lett., 103 (1989) 56-63. Nakagawa, T., Kakehata, S., Akaike, N., Komune, S., Takasaka, T. and Uemura, T., Calcium channel in isolated outer hair cells of guinea pig cochlea, Neurosci. Lett., 125 (1991) 81-84. Nakagawa, T., Kakehata, S., Akaike, N., Komune, S., Takasaka, T. and Uemura T., Effects of Ca 2+ antagonists and aminoglycoside antibiotics on Ca 2÷ current in isolated outer hair ceils of guinea pig cochlea, Brain Res., 580 (1992) 345-347. Nakai, Y., Konishi, K., Chang, K.C., Ohashi, K., Morisaki, N., Minowa, Y. and Morimoto, A., Ototoxicity of the anticancer drug cisplatin, Acta Otolaryngol. (Stockh.), 93 (1982) 227-232. Ohtani, I., Anzai, T., Aikawa, T. and Okamura, H., Toxicity of carboplatin vs. cisplatin, Practica Otol. (Kyoto), Suppl. 32 (1989) 49-55. Saito, T., Moataz, R. and Dulon, D., Cisplatin blocks depolarization-induced calcium entry in isolated cochlear outer hair cells, Hear. Res., 56 (1991) 143-147. Saito, T. and Aran, J.-M., Comaparative ototoxicity of cisplatin during acute and chronic treatment, ORL, 56 (1994) 315-320. Singh, G. and Koropatnick, J., Differential toxicity of cis and trans isomers of dichlorodiammineplatinum, J. Biochem. Toxicol., 3 (1988) 223-233. Singh, G., A possible cellular mechanism of cisplatin-induced nephrotoxicity, Toxicology, 58 (1989) 71-80. Takada, A. and Schacht J., Calcium antagonism and reversibility of gentamicin-induced loss of cochlear microphonics in the guinea pig, Hear. Res., 8 (1982) 179-186. Yamamoto, T., Kakehata, S., Saito, T., Saito, H. and Akaike, N., Cisplatin blocks voltage-dependent calcium current in dissociated outer hair cells of guinea-pig cochlea, Brain Res., 648 (1994) 296-298.
Brain Research 697 (1995) 276-279
Short communication
Transplatin blocks voltage-dependent calcium current in isolated cochlear outer hair cells but is not ototoxic in vivo Takehisa Saito *, Takehito Yamamoto, Zhi Jian Zhang, Takechiyo Yamada, Yasuhiro Manabe, Hitoshi Saito Department of Otolaryngology, Fukui Medical School, Matsuoka-cho, Yoshida-gun, 910-11 Fukui, Japan Accepted 25 July 1995
Abstract
The effects of cisplatin (cis-DDP) and transplatin (trans-DDP) on the voltage-dependent calcium current (/ca) of outer hair cells isolated from the guinea pig cochlea were compared using the whole-cell patch-clamp technique. Both cis-DDP and trans-DDP blocked Ica in a concentration-dependent manner. Trans-DDP did not show ototoxicity or nephrotoxicity in vivo. From these results, it was suggested that the effect of cis-DDP on Ica does not directly lead to cell death. Keywords: Transplatin; Cisplatin; Ototoxicity; Cochlear outer hair cell; Whole-cell patch-clamp; Voltage-dependent calcium channel; Compound action potential
The precise ototoxic mechanism of cisplatin has not yet been demonstrated. As one of the acute ototoxic mechanisms, blocking of the calcium channel [11,16] or mechano-electric transduction channel [4] of the inner ear sensory hair cells in vitro has been reported. These blocking actions on the calcium channel may affect the first step of sound perception system in the cochlea and produce functional disorders [5]. There are many drugs, however, which can affect the voltage-dependent calcium channel, such as calcium channel blockers and heavy metals [8], and some of these do not induce ototoxicity in vivo. Although cisplatin has potent ototoxicity in vivo [9,12], cisplatin did not reduce outer hair cell (OHC) viability in vitro within 6 h after incubation as previously reported [11]. It was previously reported that trans-isomer transplatin has no anti-tumor sensitivity or nephrotoxicity [13]. In the present study, blocking action on the voltage-dependent calcium channel in vitro and the ototoxicity and nephrotoxicity in vivo were compared in cisplatin and transplatin. The toxicities of cis- and transplatin in vivo and in vitro were compared to verify whether these blocking actions at the cell membrane level could lead to chronic toxicity or cell death. Albino guinea pigs (250-400 g) exibiting normal Preyer
* Corresponding author. Fax: (81) (776) 61-8118. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 9 9 8 - 1
reflex were used. The auditory bullae were removed after decapitation and strips of the organ of Corti were collected. The preparation of OHCs was treated enzymatically in Krebs solution containing 500 protease U / m l of dispase (Godo Shusei) for 30-60 s at room temperature. The OHCs in the second and third turns were dissociated mechanically by gentle pipetting. Whole-cell recordings with patch-clamp pipettes were performed as previously described [2]. The external solution contained choline-C1 140 mM, CsC1 5 mM, CaC12 10 mM, HEPES 10 mM and glucose 140 mM. The internal solution contained N-methyl-o-glucamine(NMG)-C1 30 mM, NMG-aspartate 90 mM, TEA-C1 30 mM, CaC12 0.5 mM, MgC12 5 mM, EGTA 5 mM, ATP (disodium salt) 5 mM, cAMP 0.2 mM, GTP (sodium salt) 1 mM and HEPES 10 mM. The pH of external and internal solutions was adjusted to 7.4 and 7.2 with Tris-OH respectively. The voltage-dependent K + currents were blocked by a pipette solution containing TEA + and NMG + instead of K +. Leakage current was subtracted by multiplying the current evoked by - 10 mV voltage command. Cisplatin (cis-DDP or cis-platinum (II) diammine dichloride, Sigma) and transplatin (trans-DDP or trans-platinum (II) diammine dichloride, Sigma) were dissolved in an external solution just before use. Drugs were applied by the use of Y-tube method, by which the solution surrounding a dissociated cell could be completely exchanged within 20 ms [6].
T. Saito et al. / Brain Research 697 (1995) 276-279
Calcium current amplitude was measured before and after the application of cis-DDP or trans-DDP. Twenty-two animals were divided into 3 groups and administered cis-DDP or trans-DDP as follows. Group 1 consisted of 8 guinea pigs treated intramuscularly with 7.5 m g / k g of cis-DDP at an interval of 5 days (total 15 m g / k g ) , group 2 consisted of 9 animals treated with 30 m g / k g of transplatin (total 60 m g / k g ) , and group 3 (control group) consisted of 5 animals treated with 7.5 m l / k g of saline. From the preliminary experiment, 7.5 m g / k g of trans-DDP had not caused functional or morphological damage to the cochlea or kidney (data not shown). Two days after the final administration, guinea pigs were anesthetized with pentobarbital. They underwent tracheostomy and were ventilated with a respirator. The right cochlea was then exposed by a submandibular approach and a needle electrode was placed on the bony surface of the cochlea near the round window. N 1 thresholds of compound action potential (CAP) were measured using filtered clicks at 4 and 8 kHz. After functional observation, cochleae were fixed by 1% osmium tetroxide (0.1 M phosphate buffer, pH7.4) for 1 h. After the surface preparation, 50 OHCs in each row in the middle part of each turn of the cochlea were counted and cochleograms were made. In other groups (control group (n = 5), cis-DDP group (n = 5) and trans-DDP group (n = 8)), serum was taken from the heart and blood urea nitrogen (BUN) and creatinine levels were measured to assess renal function. Analysis of the significance of differences was calculated by Student's t-test. The inhibitory actions of cis-DDP and trans-DDP progressed time-dependently and it took 3 to 4 min to reach a steady state. After washing out the drugs, the current amplitude gradually recovered toward the control level. Holding potential (V H) was - 7 0 mV. Fig. 1 shows the current-voltage relationships of the Ca 2÷ current (/ca) in the presence and absence of 10 -3 M trans-DDP. Current
MP(m~O
.
o
so
VN
I~
lcontrol r t t m ' v v p 10"3M -100
I(pA) Fig. 1. Effect of trans-DDP on the Ca 2+ current (/Ca)' Holding potential (V H) was - 7 0 mV. I - V relationships of /Ca in the presence or absence of 10 -3 M trans-DDP indicated the inhibitory effect of trans-DDP on /Ca in cochlear outer hair cells of the guinea pig.
277
1.0 0.9
g .-i t~
0.8
(o oisov
o
i
0.7
LOIra~-DDP~
0.6 0.5
I
10 -s
|
i
,
m
. . . .
i
.
.
.
.
.
.
.
.
10 -4
i
10 -3
Concentration of antagonists (M) Fig. 2. Concentration-inhibition curves of antagonists for/ca. Each point is the average of 4 to 7 cells and vertical bars represent + S.E.M.
amplitude was measured after the application of the drug for 4 min, at which time a steady state of inhibition had been established. It has already been reported that cis-DDP blocked the voltage-dependent Ca 2÷ channel [16] and this Ca z+ channel in OHCs of guinea pig was of the L-type [7]. In the present study, trans-DDP also blocked voltage-dependent Ca 2÷ channel and reversibly suppressed the/ca to almost the same degree as cis-DDP. That is to say that cisand trans-DDP acted as Ca 2÷ channel antagonists and reduced the amplitudes of the /ca" Fig. 2 shows the concentration-inhibition curves of cis- and trans-DDP on the /Ca. Both of these drugs suppressed the /Ca in a concentration-dependent manner. The threshold concentration of these drugs depressing the /Ca was 10 -4 M. The N 1 thresholds of CAP in animals treated with cis-DDP was significantly elevated to 80 dB (4 kHz: 58 _+ 16.1 dB ( P < 0.001), 8 kHz: 67.6 _ 20.6 dB ( P < 0.001)). However, animals treated with trans-DDP showed almost the same thresholds (4 kHz: 12 _+ 3.0 dB, 8 kHz: 20 _+ 3.2 dB) as the control group (4 kHz: 13 _+ 3.8 dB, 8 kHz: 23 +_ 3.6 dB) (Fig. 3), suggesting that trans-DDP did not affect hearing in the guinea pig. Fig. 4 shows the cochleograms of the animals exposed to cis-DDP and trans-DDP. In the cis-DDP group, the viabilities of OHCs were significantly decreased in all four tums of the cochlea compared with those in the trans-DDP group. Viability in the basal turn was 63 _+ 18.5%. These morphological alterations appeared to concur with the electrophysiological data. BUN and creatinine levels ( m g / d l ) in serum were significantly elevated in the cis-DDP group (BUN; 115.1 _+ 9.2, Creatinine; 1.9 _+ 0.7) compared with those in the control group (BUN; 19.9 _+ 1.2, Creatinine; 0.3 +_ 0.1). In the trans-DDP group, BUN level (19.9 _+ 1.2) showed no significant difference compared with the control group, whereas the creatinine level was slightly increased (0.4 _ 0.1 ( P < 0.05)). From the present study, it has been clarified that transDDP also blocked voltage-dependent /Ca to almost the same degree as cis-DDP. This blocking action has also
T. Saito et al. / Brain Research 697 (1995) 276-279
278
been induced by aminoglycoside antibiotics such as neomycin, gentamicin, kanamycin, and streptomycin at a high concentration of 1 mM [8] as same concentration as trans-DDP observed in this study. Furthermore, Ca 2+ channel blockers and heavy metals blocked /Ca [8]. These Ca 2÷ channel antagonists block the influx of Ca 2+ through the L-type Ca 2÷ channel in OHCs, resulting in a reduction of [Ca 2+ ]i elevation. Consequently, the Ca2+-activated K ÷ channel was suppressed and the amplitude of microphonic potential decreased concomitantly with a depolarization of the membrane potential [8]. However, in vivo experiment is necessary to support this theory. Because these observations were performed in vitro using isolated outer hair cells of the guinea pig cochlea, the environment around the cells and the condition of the cell after isolation are extremely different from those in vivo. Concerning in vivo experiment using the perilymphatic perfusion method, Ca 2+ channel blocker, nimodipine, has reduced the cochlear microphonic potential (CM) and negative summating potential ( - S P ) [1]. In addition, perilymphatic perfusion of gentamicin (GM) lowered the magnitude of CM [15] and cis-DDP caused a large loss of N I threshold sensitivity of auditory brainstem responses (ABRs) [5]. McAlpine and Jonestone [5] suggested that the basolateral walls of the hair cells, the synapses and the neural elements themselves, which were in close proximity to the perilymph of the scala tympani, were not directly affected by cis-DDP. Because a high concentration of cis-DDP or GM (3 mM) was required in scala tympani perfusion to cause any significant hearing losses, whereas scala media iontophoresis of 5 /zM cis-DDP caused a immediate loss of sensitivity in the N 1 response. Therefore, they considered that N 1 threshold elevation was caused
dB SPL 80-:
41fflz
&
60. 402o-
v
J~
o SPL. 80.
8Xnz
l
60,
4o$
2o-
•
i: ciJ-I~t~ nwm-Dll~
Fig. 3. The N 1 thresholds of C A P (4 and 8 k H z filtered clicks) after treatments with c i s - D D P and trans-DDP. The thresholds in the c i s - D D P g r o u p w e r e significantly elevated c o m p a r e d with those in the control and t r a n s - D D P groups.
(%) 10~
©
6(3
'~ •~
413
[ ] Cis-DDP • Trans-DDP • p < 0.05 ** p < 0.0l
20 0 T1
T2
T3
T4
Fig. 4. The c o c h l e o g r a m s after treatments with c i s - D D P and trans-DDP. In the c i s - D D P group, the viabilities of O H C s were significantly decreased in all four turns c o m p a r e d with those in the t r a n s - D D P group. T1 = Basal turn; T2 = second turn; T3 = third turn; T 4 - apical turn. Vertical bars represent + S.D.
by blocking the mechano-electrical transduction (MET) channel on the apical surface of the OHCs. In this context, Kimitsuki et al. [4] reported that cis-DDP blocked MET channel in isolated chick cochlear hair cell at a concentration of 1 mM using a whole-cell patch-clamp technique. This concentration was higher by three orders of magnitude than that concluded under the experiment by scala media iontophoresis. For this reason, it has been explained that the potential across the apical membrane of the hair cell in vivo was much higher than that in vitro because of the existence of the endocochlear potential, and higher concentration of cis-DDP might be necessary for the drug to act on the MET channel in isolated hair cells. Thus, it is very difficult to extrapolate in vitro and in vivo observations. The perilymphatic concentration of platinum was 5 /xM even after a single intramuscular administration with large dose of 10 m g / k g cis-DDP [10]. This value was lower by three orders of magnitude than the concentration necessary to reduce the CM by scala tympani perfusion in vivo or to block the voltage-dependent Ca 2÷ channel and MET channel in vitro. These data indicate that a very high dose of cis-DDP by general administration is necessary to affect these channels in vivo. If platinum compounds exert their toxic effect solely as heavy metals, trans-DDP should be similar in toxicity to cis-DDP. This hypothesis was not supported by our functional and morphological studies. Karen et al. [3] have reported that platinum values in organs such as the kidney, liver, and small intestine after trans-DDP treatment were much higher than those after treatment with cis-DDP. The perilymphatic concentrations of platinum after general administration of equal doses of cis-DDP and trans-DDP were proven to be almost the same by our preliminary study (data not shown). However, trans-DDP did not have ototoxicity in vivo even after the administration of a high dose (30 m g / k g ) . Trans-DDP, administered at total doses two- to four-fold (34 ~ 56 m g / k g ) those of cis-DDP, was also non-nephrotoxic on histological and functional assess-
T. Saito et al. / B r a i n Research 697 (1995) 276-279
ment [3]. Regarding nephrotoxicity, it has been suggested that some form of sterical interaction of cis-DDP with cellular constituents, which differed from that of transDDP, was essential for toxicity [3]. Candidates for this interaction might include DNA, RNA or proteins. Recent studies suggested that mitochondrial DNA in the renal cell might be affected by cis-DDP [14], although it has not yet been clarified whether this effect applied to cochlear hair cells. In conclusion, trans-DDP blocked voltage-dependent /Ca to almost the same degree as cis-DDP using a whole-cell patch-clamp method. However, trans-DDP did not show ototoxicity or nephrotoxicity in vivo, suggesting that the effect on the voltage-dependent Ca 2+ channel by cis-DDP does not directly cause cell death. Further investigation will be needed to provide more insight into the mechanisms of ototoxicity by cis-DDP.
References
[5] [6]
[7]
[8]
[9]
[10]
[11]
[12] [13]
[1] Bobbin, R.P., Jadtreboff, P.J., Fallon, M. and Littman, T., Nimodipine, an L-channel antagonist, reverses the negative summating potential recorded from the guinea pig cochlea, Hear. Res., 46 (1990) 277-288. [2] Kakehata, S., Nakagawa, T., Takasaka T. and Akaike, N., Cellular mechanism of acetylcholine-induced response in dissociated outer hair cells of guinea-pig cochlea, J. Physiol., 463 (1993) 227-244. [3] Karen, S.B., Harrington D.A., Long, D.A. and Jackson J.E., Relative lack of toxicity of transplatin compared with cisplatin in rodants, J. Comp. Pathol., 105 (1991) 367-375. [4] Kimitsuki, T., Nakagawa, T., Hisashi, K., Komune, S. and
[14] [15]
[16]
279
Komiyama S., Cisplatin blocks mechano-electric transducer current in chick cochlear hair cells, Hear. Res., 71 (1993) 64-68. McAlpine, D. and Johnstone B.M., The ototoxic mechanism of cisplatin, Hear. Res., 47 (1991) 191-204. Murase, K., Ryu, P.D. and Randic, M., Excitatory and inhibitory amino acids- and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons, Neurosci. Lett., 103 (1989) 56-63. Nakagawa, T., Kakehata, S., Akaike, N., Komune, S., Takasaka, T. and Uemura, T., Calcium channel in isolated outer hair cells of guinea pig cochlea, Neurosci. Lett., 125 (1991) 81-84. Nakagawa, T., Kakehata, S., Akaike, N., Komune, S., Takasaka, T. and Uemura T., Effects of Ca 2+ antagonists and aminoglycoside antibiotics on Ca 2÷ current in isolated outer hair ceils of guinea pig cochlea, Brain Res., 580 (1992) 345-347. Nakai, Y., Konishi, K., Chang, K.C., Ohashi, K., Morisaki, N., Minowa, Y. and Morimoto, A., Ototoxicity of the anticancer drug cisplatin, Acta Otolaryngol. (Stockh.), 93 (1982) 227-232. Ohtani, I., Anzai, T., Aikawa, T. and Okamura, H., Toxicity of carboplatin vs. cisplatin, Practica Otol. (Kyoto), Suppl. 32 (1989) 49-55. Saito, T., Moataz, R. and Dulon, D., Cisplatin blocks depolarization-induced calcium entry in isolated cochlear outer hair cells, Hear. Res., 56 (1991) 143-147. Saito, T. and Aran, J.-M., Comaparative ototoxicity of cisplatin during acute and chronic treatment, ORL, 56 (1994) 315-320. Singh, G. and Koropatnick, J., Differential toxicity of cis and trans isomers of dichlorodiammineplatinum, J. Biochem. Toxicol., 3 (1988) 223-233. Singh, G., A possible cellular mechanism of cisplatin-induced nephrotoxicity, Toxicology, 58 (1989) 71-80. Takada, A. and Schacht J., Calcium antagonism and reversibility of gentamicin-induced loss of cochlear microphonics in the guinea pig, Hear. Res., 8 (1982) 179-186. Yamamoto, T., Kakehata, S., Saito, T., Saito, H. and Akaike, N., Cisplatin blocks voltage-dependent calcium current in dissociated outer hair cells of guinea-pig cochlea, Brain Res., 648 (1994) 296-298.