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Prevention of cisplatin ototoxicity: Efficacy of micronized flavonoid fraction in a guinea pig model
International Journal of Pediatric Otorhinolaryngology 76 (2012) 1343–1346
Contents lists available at SciVerse ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Prevention of cisplatin ototoxicity: Efficacy of micronized flavonoid fraction in a guinea pig model M. Sinan Bas¸og˘lu, Erdem Eren *, Hale Aslan, Aslıhan Gu¨rcan Bingo¨lballı, ¨ ztu¨rkcan, Hu¨seyin Katılmıs¸ Sedat O Department of Otolaryngology Head & Neck Surgery, Atatu¨rk Research and Education Hospital, I˙zmir, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 March 2012 Received in revised form 29 May 2012 Accepted 3 June 2012 Available online 3 July 2012
Objective: To evaluate the effectiveness of micronized flavonoid fraction in preventing cisplatin ototoxicity in a guinea pig model. Methods: This study was conducted on 23 guinea pigs in the Animal Laboratory of Izmir Atatu¨rk Training and Research Hospital. Animals were divided into three groups: Group 1 consisted of eight animals receiving cisplatin only; Group 2 contained eight animals receiving cisplatin + micronized flavonoid fraction; and Group 3 contained seven animals that received micronized flavonoid fraction only. Their cochlear reserve was evaluated by measuring the distortion product otoacoustic emission on days 0 and 7. Results: In Groups 1 and 2, the intragroup signal–noise ratios were statistically different at all frequencies tested (based on negative ranks, p < 0.05). In Group 3, the intragroup signal–noise ratios did not differ significantly at the frequencies tested (p > 0.05). Comparison of the three groups showed statistically significant differences among the groups (p < 0.05). The post hoc Bonferroni correction showed statistically significant differences among all three groups (p < 0.016). The median signal–noise ratio of the three groups tended to increase (z-value was positive for all the frequencies tested; p < 0.01). Conclusion: Micronized flavonoid fraction (Daflon) is effective against cisplatin-induced ototoxicity in guinea pigs. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ototoxicity Micronized flavonoid fraction Cisplatin Distortion product otoacoustic emissions
1. Introduction Cisplatin is widely used in the chemotherapeutic treatment of various malignancies, including testicular, ovarian, bladder, cervical, head and neck, and non-small-cell lung cancers. The use of this agent is limited by risks of nephrotoxicity, neurotoxicity, and ototoxicity. Cisplatin ototoxicity is manifested by sensorineural hearing loss, which can be severe to profound after high-dose chemotherapy [1]. Cisplatin-related hearing loss is usually bilateral and appears first at high frequencies. Progression to lower frequencies may occur with continued therapy [2]. Cisplatin has been shown to target three areas in the cochlea: the hair cells in the basal turn of the organ of Corti; the spiral ganglion cells; and the lateral wall tissues (the spiral ligament and the stria vascularis). ROS (reactive oxygen species) mimic the effects of cisplatin on outer hair cells in vitro, and cisplatin reacts with cochlear tissue explants to generate ROS. This can lead to a calcium influx within cochlear cells, resulting in apoptosis. Excessive ROS generated by cisplatin can overwhelm the antioxidant defense
* Corresponding author at: Piri Reis Mh. I˙no¨nu¨ Cd. 252/17 Konak, I˙zmir, Turkey. Tel.: +90 0232 261 93 00; fax: +90 0232 243 15 30. E-mail address: [email protected] (E. Eren). 0165-5876/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2012.06.003
mechanisms within the cochlea, activating apoptosis of outer hair cells [1]. Flavonoids have been studied for their antioxidant and radicalscavenging properties, and it has been reported that Daflon, a micronized flavonoid fraction (MPFF) consisting of 90% diosmin and 10% hesperidin, is a powerful scavenger of antioxidant enzymes such as superoxide, singlet oxygen, and hydroxyl radicals [3]. At the microcirculation level, MPFF reduces capillary hyperpermeability and increases capillary resistance by protecting microvessels from inflammatory damage. These effects may be related to the effects of MPFF on the synthesis of NO. MPFF also reduces the expression of adhesion molecules on endothelial cells (ICAM1, VCAM1) [4] and those on leukocytes (L-selectin, VLA-4, CD 11b) [5], and it inhibits the adhesion, migration, and activation of leukocytes at the capillary level. This leads to a reduction in the release of inflammatory mediators, principally oxygen-free radicals and prostaglandins (PGE2, PGF2a) [6,7]. To our knowledge, the protective effect of micronized, purified flavonoid fractions (Daflon 500 mg, Servier, France) (MPFF) in cisplatin ototoxicity has not been previously reported. In the present study, the effect of micronized, purified flavonoid fractions on cisplatin-induced ototoxicity in guinea pigs was evaluated by means of distortion product otoacoustic emissions (DPOAEs).
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2. Methods 2.1. Animals The experimental animals were 23 adult female albino guinea pigs weighing 300–400 g. After purchase, the animals were kept in the Animal Laboratory of Izmir Atatu¨rk Training and Research Hospital for 1 week. The guinea pigs had free access to water and commercial food and were housed in temperature-controlled rooms with a 12-h light/dark cycle. The study was performed in the Animal Laboratory of Izmir Atatu¨rk Training and Research Hospital. This study was approved by the Committee for Ethics Council of the Bornova Veterinary Control and Research Institute. 2.2. Anesthesia The animals were anesthetized with 30 mg/kg ketamine hydrochloride (Ketalar, Eczacibasi Ilac Sanayi ve Ticaret A.S, Luleburgaz, Turkey) and 4 mg/kg xylazine (Alfazyne 2%, Alfasan International B.V., Woerden, The Netherlands) given as an intraperitoneal infusion before testing. The depth of anesthesia was determined by the pedal reflex. To maintain anesthesia during testing, half-doses of xylazine/ ketamine were administered as needed.
Wallis and Mann–Whitney U-tests. Statistical significance was set at p < 0.05, and confidence intervals were 95%. The Mann– Whitney U-test with Bonferroni correction was used as a post hoc test when a statistically significant result was obtained. To avoid type 1 errors, statistical significance was set at p < 0.016 (Bonferroni correction). The Jonkheere–Terpstra test was used to assess whether the order of the three groups was meaningful. Statistical significance was set at p < 0.05. 3. Results The SNR values of all three groups are shown in Figs. 1–3, respectively (Fig. 1: cisplatin, Fig. 2: cisplatin + MPFF, Fig. 3: MPFF). Baseline SNR values (pre and post intervention) are presented in Table 1. In Groups 1 and 2, intragroup SNRs were significantly different at all frequencies tested (p < 0.05). In Group 3, the intragroup SNR was not significantly different across the frequencies tested (p > 0.05). Intergroup comparison of the three groups showed statistically significant differences among the groups (p < 0.05) (Fig. 4). The post hoc Bonferroni correction showed statistically significant differences among all three groups (p < 0.016). The median SNR of the three groups tended to increase (z-value was positive for all the frequencies tested; p < 0.01).
2.3. Experimental design
4. Discussion
Cisplatin-induced ototoxicity appears within hours and stabilizes over 3 days, continuing for up to 7 days of high dose cisplatin application. In our experiment, DPOAE assessment was performed on day 7.
Cisplatin ototoxicity is a frequent undesirable side effect of cisplatin chemotherapy. Survivors of cancer treatment with cisplatin, particularly children, may suffer severe and life-long 35
Group 1 – Eight animals (cisplatin only). Cisplatin 13 mg/kg (Cisplatin DBL, Faulding Pharmaceuticals, Warwickshire, UK) was slowly infused intraperitoneally. Group 2 – Eight animals (cisplatin and MPFF). Cisplatin 13 mg/kg was injected intraperitoneally as a slow infusion, and Daflon 500 mg (90% Diosmin, 10% hesperidin) was administered orogastrically at 100 mg/kg/day for 7 days. Group 3 – Seven animals (MPFF only). Daflon 500 mg (S 5682, Servier, France) was administered orogastrically at 100 mg/kg/ day for 7 days.
25 15 5 -5
Before Cisplatin
0.5kHz
1kHz
2kHz
3kHz
4kHz
5kHz
8kHz
After Cisplatin
-15 -25
2.4. OAE measurements DPOAE (Vivosonic Integry V500, Toronto, Ontario, Canada) was measured using tympanometry probes adjusted to fit in the guinea pig aural canal. Baseline DPOAE measurements were taken before the procedure. The cochlear activity in the ears of all guinea pigs was examined on days 0 and 7 using DPOAE in sedated animals. Once the probe was placed with a good seal in the canal, DPOAEs were measured. Equilevel primary tones f1 (65 dB) and f2 (55 dB) were fixed at f1/f2 = 1.22, and DPOAEs were measured at seven different frequencies ranging from 0.5 to 8 kHz (0.5, 1, 2, 3, 4, 5, and 8 kHz). By calculating the difference between the distortion products and noise standard deviations, the signal–noise ratios (SNR) for each frequency were found. Two DPOAE measurements were obtained on days 0 and 7 to detect any possible deterioration in cochlear activity. All measurements were recorded in a quiet room. 2.5. Statistical analysis SPSS 16 for Windows was used for the statistical analysis. Intragroup differences in SNR were evaluated with the Wilcoxon signed-rank test, and intergroup differences with the Kruskal–
-35
Fig. 1. SNR (signal-to-noise ratio) values in guinea pigs before and after intraperitoneal cisplatin injection.
30 20 10 Before cisplatin After Cisplatin+MPPF
0 0.5kHz
1kHz
2kHz
3kHz
4kHz
5kHz
8kHz
-10 -20 -30
Fig. 2. SNR (signal-to-noise ratio) values in guinea pigs before intraperitoneal cisplatin injection and after cisplatin injection + MPFF administration.
M. Sinan Bas¸og˘lu et al. / International Journal of Pediatric Otorhinolaryngology 76 (2012) 1343–1346
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20 30
10 20
0 10
-10
Before MPPF
1.group
After MPFF
0 0.5kHz
1kHz
2kHz
3kHz
4kHz
5kHz
2.group
-20
8kHz
3.group
-30
-10
-40
-20
-50 -30
-60
Fig. 3. SNR (signal-to-noise ratio) values in guinea pigs before and after MPFF administration.
0,5kHz
1kHz
2kHz
3kHz
4kHz
5kHz
8kHz
Fig. 4. Differences in SNR (signal-to-noise ratio) values before and after administration in each group.
disability from hearing loss. A variety of antioxidant compounds have been used to attempt to prevent apoptosis of cochlear cells. Several of these compounds contain thiol groups, which attenuate cisplatin ototoxicity, including sodium thiosulfate, diethyldithiocarbamate D or L-methionine, methylthiobenzoic acid, lipoic acid, N-acetylcysteine, tiopronin, glutathione ester, and amifostine [8]. Other agents that may function as free-radical scavengers and protect the cochlea from cisplatin damage and hearing loss in experimental animals include a-tocopherol (alone or in combination with tiopronin), aminoguanidine, D and L-methionine, and sodium salicylate [8]. MPFF containing diosmin and hesperidin (9:1) has been found to prevent ischemia and reperfusion-induced leukocyte adhesion in skeletal muscles [9]. Hesperidin, in combination with diosmin, shows a marked protective effect against inflammatory disorders, both in vivo and in vitro, possibly through a mechanism involving inhibition of eicosanoid synthesis and/or antioxidant free-radical scavenging activity [10]. Lonchampt et al. studied the scavenging properties of Daflon 500 mg on active oxygen radicals in vitro and in vivo, implying its pharmacological action on capillary hyperpermeability as well as anti-inflammatory and antiedematous actions. Daflon was shown to improve multiple aspects of acute inflammation (diapedesis of polymorphs, lymphocytes, histocytes, and macrophages) as well as those seen in chronic inflammation (new micro vascularization of the granulomatous tissues, perivascular edema, and presence of collagen fibers) [11]. In a study investigating its efficacy in the chronic treatment of inflammatory granuloma, Daflon was highly effective against edema and reduced the synthesis of PGE2 and PGF2 [12]. Cisplatin mainly affects the outer hair cells. Evoked OAEs, especially DPOAEs due to frequency specificity, are more sensitive in the evaluation of OHCs than are conventional audiometry, ultrahigh-frequency audiometry, and auditory brainstem response (ABR) [13]. In an experimental cisplatin ototoxicity model,
intratympanic dexamethasone was shown to prevent aural deficit at frequencies of 1–6 kHz as shown by DPOAE. The possible protective effects of dexamethasone as a prophylactic agent were investigated. No hearing loss was reported after dexamethasone use [14]. Erdem et al. evaluated the preventive effect of the antioxidant agent resveratrol on cisplatin-induced ototoxicity. Statistically significant reductions in DP-gram amplitudes were noted at frequencies of 1418, 2003, 3363, 5660, 8003, and 9515 Hz in the cisplatin group, strongly suggesting cisplatin-related ototoxicity. In the cisplatin and resveratrol combination group, a statistically significant difference was found between days 1 and 5 at 3996 Hz only [15]. In guinea pigs, transtympanic administration of NAC and lactated ringers solution preserved DPOAE [16]. In a study evaluating the prevention of cisplatin ototoxicity using pomegranate extract, the authors reported significant otoprotection at frequencies of 3, 4, 6, and 8 kHz as assessed by DPOAE [17]. In this study, only the cisplatin group showed significant deterioration as demonstrated by DPOAE; DPOAE did not change after MPPF administration. In the MPPF + cisplatin group, DPOAE deteriorated, but intergroup SNR values showed statistically significant differences (p < 0.016). The deterioration of SNR levels observed in the MPPF + cisplatin group may be explained by fact that MPPF did not precede the administration of cisplatin. Our results indicate that MPPF is effective against cisplatin ototoxicity. There are certain limitations of our study. Scanning electron microscopic assessment could be used to validate our results. There is an increasing trend toward intratympanic administration of drugs to prevent ototoxicity. Although we believe that oral administration of drugs such as MPPF is more feasible and less invasive, studies comparing the efficacy of the route of administration must be conducted. Additionally, further studies are needed to ensure that anti-ototoxicity drugs do not decrease the efficacy of the chemotherapeutic agent.
Table 1 SNR (signal-to-noise ratio) values of each group in guinea pigs before the intervention. Frequency
0.5 kHz 1 kHz 2 kHz 3 kHz 4 kHz 5 kHz 8 kHz
Group 1 (cisplatin only)
Group 2 (cisplatin + MPFF)
Group 3 (MPFF only)
Mean value
Standard deviation
Mean value
Standard deviation
Mean value
Standard deviation
3.14 10.03 14.42 13.07 16.08 15.23 24.42
2.49 1.68 1.50 0.78 1.81 1.93 4.28
3.92 11.19 12.04 16.59 18.74 22.57 26.86
0.86 1.53 1.36 1.32 1.41 1.85 3.05
3.90 11.28 16.74 15.26 15.94 24.07 30.07
0.56 1.29 0.88 1.12 2.56 5.43 6.19
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Micronized flavonoid fraction (Daflon1), a commercially available and safe drug, provides protection in ototoxicity of cisplatin in guinea pigs. However, further studies with different dosages and administration routes are needed to fully elucidate the effect of this drug on ototoxicity before the treatment is approved for human use. Author contributions All authors have contributed to conception and design, acquisition, analysis and interpretation of data, drafting the article or revising it critically for intellectual content and final approval of the version to be published. Conflict of interest None declared. References [1] L.P. Rybak, C.A. Whitworth, D. Mukherjea, V. Ramkumar, Mechanisms of cisplatin induced ototoxicity and prevention, Hear. Res. 226 (2007) 157–167. [2] L.P. Rybak, V. Ramkumar, Ototoxicity, Kidney Int. 72 (October (8)) (2007) 931– 935. [3] M.S. Brown, R.B. Buchanan, S.J. Karran, Clinical observations on the effects of elemental diet supplementation during irradiation, Clin. Radiol. 31 (January (1)) (1980) 19–20. [4] S.S. Shoab, J. Porter, J.H. Scurr, P.D. Coleridge-Smith, Endothelial activation response to oral micronized flavonoid therapy in patients with chronic venous disease. A prospective study, Eur. J. Vasc. Endovasc. Surg. 17 (1999) 313. [5] M. Cospite, V. Cospite, Treatment of hemorrhoids with DAFLON 500 mg, Phlebology 7 (Suppl. 2) (1992) 53–56.
[6] B. Friesenecker, A.G. Tsai, C. Allegra, M. Intaglietta, Oral administration of purified micronized flavonoid fraction suppresses leukocyte adhesion in ischemia–reperfusion injury: in vivo observations in the hamster skin fold, Int. J. Microcirc. 14 (1994) 50–55. [7] M. Longchampt, Protective effect of purified flavonoid fraction against reactive oxygen radicals. In vivo and in vitro study, Arzneimittelforschung/Drug Res. 39 (1989) 882–885. [8] L.P. Rybak, C.A. Whitworth, Ototoxicity: therapeutic opportunities, Drug Discov. Today 10 (2005) 1313–1321. [9] R.J. Korthuis, D.C. Gute, Adhesion molecule expression in postischemic microvascular dysfunction: activity of a micronized purified flavonoid fraction, J. Vasc. Res. 36 (Suppl. 1) (1999) 15–23. [10] T. Jean, M.C. Bodinier, Mediators involved in inflammation: effects of Daflon 500 mg on their release, Angiology 45 (June (6 Pt 2)) (1994) 554–559. [11] M. Lonchampt, B. Guardiola, N. Sicot, M. Bertrand, L. Perdrix, J. Duhault, Protective effect of a purified flavonoid fraction against reactive oxygen radicals. In vivo and in vitro study, Arzneimittelforschung 39 (August (8)) (1989) 882–885. [12] M. Damon, O. Flandre, F. Michel, L. Perdrix, C. Labrid, A. Crastes de Paulet, Effect of chronic treatment with a purified flavonoid fraction on inflammatory granuloma in the rat. Study of prostaglandin E2 and F2 alpha and thromboxane B2 release and histological changes, Arzneimittelforschung 37 (October (10)) (1987) 1149– 1153. [13] B.D. Ress, K.S. Sridhar, T.J. Balkany, G.M. Waxman, B.B. Stagner, B.L. LonsburyMartin, Effects of cis-platinum chemotherapy on otoacoustic emissions: the development of an objective screening protocol. Third place – Resident Clinical Science Award 1998, Otolaryngol. Head Neck Surg. 121 (1999) 693–701. [14] A. Daldal, O. Odabasi, B. Serbetcioglu, The protective effect of intratympanic dexamethasone on cisplatin-induced ototoxicity in guinea pigs, Otolaryngol. Head Neck Surg. 137 (November (5)) (2007) 747–752. [15] T. Erdem, T. Bayindir, A. Filiz, M. Iraz, E. Selimoglu, The effect of resveratrol on the prevention of cisplatin ototoxicity, Eur. Arch. Otorhinolaryngol. (2011) [Epub ahead of print; 21 December 2011]. [16] W.T. Choe, N. Chinosornvatana, K.W. Chang, Prevention of cisplatin ototoxicity using transtympanic N-acetylcysteine and lactate, Otol. Neurotol. 25 (November (6)) (2004) 910–915. [17] A.M. Teker, V. Kahya, Z.M. Yazici, Y. Meral, O. Gedikli, G. Hafız, Protective effect of pomegranate extract on cisplatin-induced ototoxicity, Int. Adv. Otol. 6 (3) (2010) 380–385.
Contents lists available at SciVerse ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Prevention of cisplatin ototoxicity: Efficacy of micronized flavonoid fraction in a guinea pig model M. Sinan Bas¸og˘lu, Erdem Eren *, Hale Aslan, Aslıhan Gu¨rcan Bingo¨lballı, ¨ ztu¨rkcan, Hu¨seyin Katılmıs¸ Sedat O Department of Otolaryngology Head & Neck Surgery, Atatu¨rk Research and Education Hospital, I˙zmir, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 March 2012 Received in revised form 29 May 2012 Accepted 3 June 2012 Available online 3 July 2012
Objective: To evaluate the effectiveness of micronized flavonoid fraction in preventing cisplatin ototoxicity in a guinea pig model. Methods: This study was conducted on 23 guinea pigs in the Animal Laboratory of Izmir Atatu¨rk Training and Research Hospital. Animals were divided into three groups: Group 1 consisted of eight animals receiving cisplatin only; Group 2 contained eight animals receiving cisplatin + micronized flavonoid fraction; and Group 3 contained seven animals that received micronized flavonoid fraction only. Their cochlear reserve was evaluated by measuring the distortion product otoacoustic emission on days 0 and 7. Results: In Groups 1 and 2, the intragroup signal–noise ratios were statistically different at all frequencies tested (based on negative ranks, p < 0.05). In Group 3, the intragroup signal–noise ratios did not differ significantly at the frequencies tested (p > 0.05). Comparison of the three groups showed statistically significant differences among the groups (p < 0.05). The post hoc Bonferroni correction showed statistically significant differences among all three groups (p < 0.016). The median signal–noise ratio of the three groups tended to increase (z-value was positive for all the frequencies tested; p < 0.01). Conclusion: Micronized flavonoid fraction (Daflon) is effective against cisplatin-induced ototoxicity in guinea pigs. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ototoxicity Micronized flavonoid fraction Cisplatin Distortion product otoacoustic emissions
1. Introduction Cisplatin is widely used in the chemotherapeutic treatment of various malignancies, including testicular, ovarian, bladder, cervical, head and neck, and non-small-cell lung cancers. The use of this agent is limited by risks of nephrotoxicity, neurotoxicity, and ototoxicity. Cisplatin ototoxicity is manifested by sensorineural hearing loss, which can be severe to profound after high-dose chemotherapy [1]. Cisplatin-related hearing loss is usually bilateral and appears first at high frequencies. Progression to lower frequencies may occur with continued therapy [2]. Cisplatin has been shown to target three areas in the cochlea: the hair cells in the basal turn of the organ of Corti; the spiral ganglion cells; and the lateral wall tissues (the spiral ligament and the stria vascularis). ROS (reactive oxygen species) mimic the effects of cisplatin on outer hair cells in vitro, and cisplatin reacts with cochlear tissue explants to generate ROS. This can lead to a calcium influx within cochlear cells, resulting in apoptosis. Excessive ROS generated by cisplatin can overwhelm the antioxidant defense
* Corresponding author at: Piri Reis Mh. I˙no¨nu¨ Cd. 252/17 Konak, I˙zmir, Turkey. Tel.: +90 0232 261 93 00; fax: +90 0232 243 15 30. E-mail address: [email protected] (E. Eren). 0165-5876/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2012.06.003
mechanisms within the cochlea, activating apoptosis of outer hair cells [1]. Flavonoids have been studied for their antioxidant and radicalscavenging properties, and it has been reported that Daflon, a micronized flavonoid fraction (MPFF) consisting of 90% diosmin and 10% hesperidin, is a powerful scavenger of antioxidant enzymes such as superoxide, singlet oxygen, and hydroxyl radicals [3]. At the microcirculation level, MPFF reduces capillary hyperpermeability and increases capillary resistance by protecting microvessels from inflammatory damage. These effects may be related to the effects of MPFF on the synthesis of NO. MPFF also reduces the expression of adhesion molecules on endothelial cells (ICAM1, VCAM1) [4] and those on leukocytes (L-selectin, VLA-4, CD 11b) [5], and it inhibits the adhesion, migration, and activation of leukocytes at the capillary level. This leads to a reduction in the release of inflammatory mediators, principally oxygen-free radicals and prostaglandins (PGE2, PGF2a) [6,7]. To our knowledge, the protective effect of micronized, purified flavonoid fractions (Daflon 500 mg, Servier, France) (MPFF) in cisplatin ototoxicity has not been previously reported. In the present study, the effect of micronized, purified flavonoid fractions on cisplatin-induced ototoxicity in guinea pigs was evaluated by means of distortion product otoacoustic emissions (DPOAEs).
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2. Methods 2.1. Animals The experimental animals were 23 adult female albino guinea pigs weighing 300–400 g. After purchase, the animals were kept in the Animal Laboratory of Izmir Atatu¨rk Training and Research Hospital for 1 week. The guinea pigs had free access to water and commercial food and were housed in temperature-controlled rooms with a 12-h light/dark cycle. The study was performed in the Animal Laboratory of Izmir Atatu¨rk Training and Research Hospital. This study was approved by the Committee for Ethics Council of the Bornova Veterinary Control and Research Institute. 2.2. Anesthesia The animals were anesthetized with 30 mg/kg ketamine hydrochloride (Ketalar, Eczacibasi Ilac Sanayi ve Ticaret A.S, Luleburgaz, Turkey) and 4 mg/kg xylazine (Alfazyne 2%, Alfasan International B.V., Woerden, The Netherlands) given as an intraperitoneal infusion before testing. The depth of anesthesia was determined by the pedal reflex. To maintain anesthesia during testing, half-doses of xylazine/ ketamine were administered as needed.
Wallis and Mann–Whitney U-tests. Statistical significance was set at p < 0.05, and confidence intervals were 95%. The Mann– Whitney U-test with Bonferroni correction was used as a post hoc test when a statistically significant result was obtained. To avoid type 1 errors, statistical significance was set at p < 0.016 (Bonferroni correction). The Jonkheere–Terpstra test was used to assess whether the order of the three groups was meaningful. Statistical significance was set at p < 0.05. 3. Results The SNR values of all three groups are shown in Figs. 1–3, respectively (Fig. 1: cisplatin, Fig. 2: cisplatin + MPFF, Fig. 3: MPFF). Baseline SNR values (pre and post intervention) are presented in Table 1. In Groups 1 and 2, intragroup SNRs were significantly different at all frequencies tested (p < 0.05). In Group 3, the intragroup SNR was not significantly different across the frequencies tested (p > 0.05). Intergroup comparison of the three groups showed statistically significant differences among the groups (p < 0.05) (Fig. 4). The post hoc Bonferroni correction showed statistically significant differences among all three groups (p < 0.016). The median SNR of the three groups tended to increase (z-value was positive for all the frequencies tested; p < 0.01).
2.3. Experimental design
4. Discussion
Cisplatin-induced ototoxicity appears within hours and stabilizes over 3 days, continuing for up to 7 days of high dose cisplatin application. In our experiment, DPOAE assessment was performed on day 7.
Cisplatin ototoxicity is a frequent undesirable side effect of cisplatin chemotherapy. Survivors of cancer treatment with cisplatin, particularly children, may suffer severe and life-long 35
Group 1 – Eight animals (cisplatin only). Cisplatin 13 mg/kg (Cisplatin DBL, Faulding Pharmaceuticals, Warwickshire, UK) was slowly infused intraperitoneally. Group 2 – Eight animals (cisplatin and MPFF). Cisplatin 13 mg/kg was injected intraperitoneally as a slow infusion, and Daflon 500 mg (90% Diosmin, 10% hesperidin) was administered orogastrically at 100 mg/kg/day for 7 days. Group 3 – Seven animals (MPFF only). Daflon 500 mg (S 5682, Servier, France) was administered orogastrically at 100 mg/kg/ day for 7 days.
25 15 5 -5
Before Cisplatin
0.5kHz
1kHz
2kHz
3kHz
4kHz
5kHz
8kHz
After Cisplatin
-15 -25
2.4. OAE measurements DPOAE (Vivosonic Integry V500, Toronto, Ontario, Canada) was measured using tympanometry probes adjusted to fit in the guinea pig aural canal. Baseline DPOAE measurements were taken before the procedure. The cochlear activity in the ears of all guinea pigs was examined on days 0 and 7 using DPOAE in sedated animals. Once the probe was placed with a good seal in the canal, DPOAEs were measured. Equilevel primary tones f1 (65 dB) and f2 (55 dB) were fixed at f1/f2 = 1.22, and DPOAEs were measured at seven different frequencies ranging from 0.5 to 8 kHz (0.5, 1, 2, 3, 4, 5, and 8 kHz). By calculating the difference between the distortion products and noise standard deviations, the signal–noise ratios (SNR) for each frequency were found. Two DPOAE measurements were obtained on days 0 and 7 to detect any possible deterioration in cochlear activity. All measurements were recorded in a quiet room. 2.5. Statistical analysis SPSS 16 for Windows was used for the statistical analysis. Intragroup differences in SNR were evaluated with the Wilcoxon signed-rank test, and intergroup differences with the Kruskal–
-35
Fig. 1. SNR (signal-to-noise ratio) values in guinea pigs before and after intraperitoneal cisplatin injection.
30 20 10 Before cisplatin After Cisplatin+MPPF
0 0.5kHz
1kHz
2kHz
3kHz
4kHz
5kHz
8kHz
-10 -20 -30
Fig. 2. SNR (signal-to-noise ratio) values in guinea pigs before intraperitoneal cisplatin injection and after cisplatin injection + MPFF administration.
M. Sinan Bas¸og˘lu et al. / International Journal of Pediatric Otorhinolaryngology 76 (2012) 1343–1346
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20 30
10 20
0 10
-10
Before MPPF
1.group
After MPFF
0 0.5kHz
1kHz
2kHz
3kHz
4kHz
5kHz
2.group
-20
8kHz
3.group
-30
-10
-40
-20
-50 -30
-60
Fig. 3. SNR (signal-to-noise ratio) values in guinea pigs before and after MPFF administration.
0,5kHz
1kHz
2kHz
3kHz
4kHz
5kHz
8kHz
Fig. 4. Differences in SNR (signal-to-noise ratio) values before and after administration in each group.
disability from hearing loss. A variety of antioxidant compounds have been used to attempt to prevent apoptosis of cochlear cells. Several of these compounds contain thiol groups, which attenuate cisplatin ototoxicity, including sodium thiosulfate, diethyldithiocarbamate D or L-methionine, methylthiobenzoic acid, lipoic acid, N-acetylcysteine, tiopronin, glutathione ester, and amifostine [8]. Other agents that may function as free-radical scavengers and protect the cochlea from cisplatin damage and hearing loss in experimental animals include a-tocopherol (alone or in combination with tiopronin), aminoguanidine, D and L-methionine, and sodium salicylate [8]. MPFF containing diosmin and hesperidin (9:1) has been found to prevent ischemia and reperfusion-induced leukocyte adhesion in skeletal muscles [9]. Hesperidin, in combination with diosmin, shows a marked protective effect against inflammatory disorders, both in vivo and in vitro, possibly through a mechanism involving inhibition of eicosanoid synthesis and/or antioxidant free-radical scavenging activity [10]. Lonchampt et al. studied the scavenging properties of Daflon 500 mg on active oxygen radicals in vitro and in vivo, implying its pharmacological action on capillary hyperpermeability as well as anti-inflammatory and antiedematous actions. Daflon was shown to improve multiple aspects of acute inflammation (diapedesis of polymorphs, lymphocytes, histocytes, and macrophages) as well as those seen in chronic inflammation (new micro vascularization of the granulomatous tissues, perivascular edema, and presence of collagen fibers) [11]. In a study investigating its efficacy in the chronic treatment of inflammatory granuloma, Daflon was highly effective against edema and reduced the synthesis of PGE2 and PGF2 [12]. Cisplatin mainly affects the outer hair cells. Evoked OAEs, especially DPOAEs due to frequency specificity, are more sensitive in the evaluation of OHCs than are conventional audiometry, ultrahigh-frequency audiometry, and auditory brainstem response (ABR) [13]. In an experimental cisplatin ototoxicity model,
intratympanic dexamethasone was shown to prevent aural deficit at frequencies of 1–6 kHz as shown by DPOAE. The possible protective effects of dexamethasone as a prophylactic agent were investigated. No hearing loss was reported after dexamethasone use [14]. Erdem et al. evaluated the preventive effect of the antioxidant agent resveratrol on cisplatin-induced ototoxicity. Statistically significant reductions in DP-gram amplitudes were noted at frequencies of 1418, 2003, 3363, 5660, 8003, and 9515 Hz in the cisplatin group, strongly suggesting cisplatin-related ototoxicity. In the cisplatin and resveratrol combination group, a statistically significant difference was found between days 1 and 5 at 3996 Hz only [15]. In guinea pigs, transtympanic administration of NAC and lactated ringers solution preserved DPOAE [16]. In a study evaluating the prevention of cisplatin ototoxicity using pomegranate extract, the authors reported significant otoprotection at frequencies of 3, 4, 6, and 8 kHz as assessed by DPOAE [17]. In this study, only the cisplatin group showed significant deterioration as demonstrated by DPOAE; DPOAE did not change after MPPF administration. In the MPPF + cisplatin group, DPOAE deteriorated, but intergroup SNR values showed statistically significant differences (p < 0.016). The deterioration of SNR levels observed in the MPPF + cisplatin group may be explained by fact that MPPF did not precede the administration of cisplatin. Our results indicate that MPPF is effective against cisplatin ototoxicity. There are certain limitations of our study. Scanning electron microscopic assessment could be used to validate our results. There is an increasing trend toward intratympanic administration of drugs to prevent ototoxicity. Although we believe that oral administration of drugs such as MPPF is more feasible and less invasive, studies comparing the efficacy of the route of administration must be conducted. Additionally, further studies are needed to ensure that anti-ototoxicity drugs do not decrease the efficacy of the chemotherapeutic agent.
Table 1 SNR (signal-to-noise ratio) values of each group in guinea pigs before the intervention. Frequency
0.5 kHz 1 kHz 2 kHz 3 kHz 4 kHz 5 kHz 8 kHz
Group 1 (cisplatin only)
Group 2 (cisplatin + MPFF)
Group 3 (MPFF only)
Mean value
Standard deviation
Mean value
Standard deviation
Mean value
Standard deviation
3.14 10.03 14.42 13.07 16.08 15.23 24.42
2.49 1.68 1.50 0.78 1.81 1.93 4.28
3.92 11.19 12.04 16.59 18.74 22.57 26.86
0.86 1.53 1.36 1.32 1.41 1.85 3.05
3.90 11.28 16.74 15.26 15.94 24.07 30.07
0.56 1.29 0.88 1.12 2.56 5.43 6.19
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M. Sinan Bas¸og˘lu et al. / International Journal of Pediatric Otorhinolaryngology 76 (2012) 1343–1346
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