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L-Carnitine Reduces Cochlear Damage Induced by Gamma Irradiation in Guinea Pigs
L-Carnitine Reduces Cochlear Damage Induced by Gamma Irradiation in Guinea Pigs
L-Carnitine Reduces Cochlear Damage Induced by Gamma Irradiation in Guinea Pigs
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Enver Altas,1 Mustafa Vecdi Ertekin,2 Cemal Gundogdu,3 and Elif Demirci3 Departments of 1 Otolaryngology, 2 Radiation Oncology, and 3 Pathology, School of Medicine, Ataturk University, Erzurum, Turkey Abstract. L-carnitine (LC) protects cells from peroxidative damage. In this study, we tested whether Lcarnitine (LC) prevents radiation-induced cochlear damage after total cranial irradiation (radiotherapy; RT). Male albino guinea pigs were randomly distributed in 3 groups. The Control group (n = 11) received neither LC nor irradiation, but saline solution ip and sham irradiation for 5 days. The RT group (n = 32) received saline solution ip as placebo therapy and exposure to total cranial irradiation of 33 Gy in 5 fractions of 6.6 Gy/day on 5 successive days, with a calculated (/ = 3.5) biological effective dose of fractionated irradiation equal to 60 Gy conventional fractionation. The LC + RT group (n = 36) received total cranial irradiation, plus LC (100 mg/kg/day, ip) for 5 days. The guinea pigs were killed at 4, 24, or 96 hr after the last dose of RT and the cochleas were enucleated for histopathologic examination. There was no cochlear degeneration in the control group. In the RT group, total cranial irradiation caused degeneration in stria vascularis (SV), spiral ganglion (SG), outer hair cells (OHC), and inner hair cells (IHC) of cochleas at 4, 24, and 96 hr. In the LC + RT group, LC administration reduced radiation-induced cochlear degeneration in SV and SG at 4, 24, and 96 hr, and in OHC and IHC at 24 and 96 hr (p <0.05). Thus, this study shows that L-carnitine can ameliorate radiation-induced cochlear damage in guinea pigs. Keywords: gamma irradiation, cochlear damage, L-carnitine, antioxidant, radioprotection Introduction Radiotherapy (RT) is used widely for both primary and adjuvant treatment of cancer. Despite its curative efficacy, RT causes side effects such as ototoxicity. Hearing loss due to ototoxicity may be severe enough to impair the quality of life in patients [1-4]. Numerous trials have been conducted to reduce or prevent the ototoxic side effects of RT. These studies have tested pantothenic acid, coenzyme A, D-methionine, histidine, brainderived neurotrophic factor with D-methionine, L-methionine, diethyldithiocarbamate, 4-methylthiobenzoic acid, ebselen, -lipoic acid, -tocoAddress correspondence to Dr. Enver Altas, Ataturk Universitesi, Tip Fakultesi, K.B.B. Anabilim Dali, Erzurum 25240, Turkey; tel 90 442 236 1212; fax 90 442 236-1301; e-mail enveraltas@hotmail.com.
pherol, vitamins A, B, C, E, and K, zinc-coppersuperoxide dismutase, nicotinamide, various amino acids, choline, ATP, glucuronic acid, chondroitin sulfate, corticoids, sulfhydryl compounds (eg, panthotenic acid, glutathione, sodium thiosulfate), piracetam, aminoguanidine, and L-carnitine [516]. The focus of the present study is L-carnitine (LC), which is crucial for mitochondrial energy production via -oxidation of fatty acids [17]. LC has been reported to have antiperoxidative effects in several tissues [18-22]. In addition to protecting cells from peroxidative stress that damages cell membrane, LC has a scavenger effect on reactive oxygen species (ROS) [18,23]. The present study tests the protective effects of ip administration of LC on cochlear damage induced in guinea pigs by exposure to total cranial irradiation.
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and inner hair cells (IHC)). The spiral ganglion (SG) was evaluated for cytoplasmic and nuclear condensation, nucleus and neuron loss; and the stria vascularis (SV) for edema, vacuolization, and loss of cells. Changes in the SV were scored as absent (0), mild (1), moderate (2) or severe (3); the percentages of degenerated cells in the SG, and the numbers of guinea pigs with degenerated hair cells (IHC and OHC) in the organ of Corti were estimated [24]. Statistics. Statistical analyses were made by the SPSS program (Statistical Package for Social Science; Windows version 11.0). The results are expressed as mean SD. Differences between groups were tested by the Mann-Whitney U test. Comparisons among more than 2 groups were performed by Friedman ANOVA; p <0.05 was considered significant.
Results No cochlear degeneration was observed in the Control group (Table 1, Fig. 1a,b,c). In the RT group, total cranial irradiation promoted degeneration in the SV (severe hydropic and vacuolar degeneration, Fig. 2a), in the OHC and IHC (hydropic and vacuolar degeneration, and loss of cells, Fig. 2b), and in the SG (cytoplasmic and nuclear condensation, nuclear and neuronal loss, Fig. 2c) at 4, 24, and 96 hr after last treatment (p <0.05) (Table 1). In the LC + RT group, LC administration decreased the RT-induced degeneration in the SV (mild hydropic and vacuolar degeneration, Fig. 3a) at 4, 24, and 96 hr, in the OHC and IHC (hydropic degeneration, Fig. 3b) at 24 and 96 hr, and in the SG (cytoplasmic and nuclear condensation, and occasional nuclear and neuronal loss, Fig. 3c) at 4, 24, and 96 hr after last treatment. These findings show that LC administration ameliorated the radiation-induced cochlear damage. (Table 1). Discussion The goal of radiation treatment of cancer patients is to deliver accurately measured doses of ionizing radiation to a defined tumor volume with minimal injurious effects on surrounding healthy tissue; by eliminating tumor cells, radiation treatment can enhance the quality of life and prolong the survival of cancer patients at reasonable cost [25]. Unfortunately, cochlear damage may occur during the irradiation therapy of patients with head and neck cancer [1].
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Table 1: Statistical analysis and scoring of histopathological changes in the organ of Corti, spiral ganglion, and stria vascularis of guinea pig cochleas in the Control group (sham irradiation and placebo), RT group (cranial irradiation and placebo), and LC-RT group (cranial irradiation and L-carnitine), at 4, 24, or 96 hr after the last treatment (see text for experimental details). Control Group Group: Treatment: (sham irradiation & placebo) Time after last treatment (hr): 4 Number of guinea pigs: 11 Degeneration in the stria vascularis Severity score = 0 11 Severity score = 1 0 Severity score = 2 0 Severity score = 3 0 mean 0 SD 0.0 p values b,c,d,e,f,g Degenerated cells (%) in the spiral ganglion mean 0 SD 0.0 p values b,c,d,e,f,g Number of guinea pigs with degenerated outer hair cells (OHC) in the organ of Corti Severity score = 0 11 Severity score = 1 0 Severity score = 2 0 Severity score = 3 0 mean 0 SD 0.0 p values b,c,d,e,f,g Number of guinea pigs with degenerated inner hair cells (IHC) in the organ of Corti Severity score = 0 11 Severity score = 1 0 mean 0 SD 0.0 p values b,c,d RT Group (irradiation & placebo) 4 24 96 12 10 10 0 0 7 5 3.42 0.51
a,e,f,g
LC+RT Group (irradiation & L-carnitine) 4 24 96 12 13 11 0 1 11 0 2.92 0.28 0 7 6 0 2.46 0.51 4 4 3 0 1.91 0.83
0 0 4 6 3.60 0.51
a,e,f,g
0 0 2 8 3.80 0.42
a,e,f,g
66.50 4.27
80.80 3.15
88.80 3.01
53.83 2.48
43.84 2.76
32.36 2.94
0 0 7 5 3.42 0.51
a,f,g
0 0 4 6 3.60 0.51
a,e,f,g
0 0 2 8 3.80 0.42
a,e,f,g
0 5 3 4 2.92 0.90
a,c,d,g
0 6 4 3 2.77 0.83
5 3 2 1 1.91 1.04
a,b,c,d,g a,b,c,d,e,g
7 5 1.42 0.51
a
4 6 1.60 0.51
a,f,g
3 7 1.70 0.48
a,e,f,g
9 3 1.25 0.45
d
11 2 1.15 0.37
c,d
10 1 1.09 0.30
c,d
Degeneration severity was scored as absent (0), mild (1), moderate (2). or severe (3). a p <0.05 vs Control group; b p <0.05 vs RT 4 hr group; c p <0.05 vs RT 24 hr group; d p <0.05 vs RT 96 hr group; e p <0.05 vs LC+RT 4 hr group; f p <0.05 vs LC+RT 24 hr group; g p <0.05 vs LC+RT 96 hr group.
Exposure of cells to ionizing radiation causes oxidative stresses that are associated with radiationinduced cytotoxicity [26]. Oxidative stress is important in the pathophysiology of ototoxicity [27]. Oxidative tolerance can occur as a defense against oxidative substances. Oxidative tolerance may explain why ototoxicity is limited in low dose RT [28]. Kim et al [29] evaluated histopathologically the cochlea of guinea pigs following single doses of irradiation (2, 6, 10, or 15 Gy). Outer hair cell damage appeared with irradiation 10 Gy, and
inner hair cell damage with irradiation 15 Gy. The morphologic changes in the stria vascularis were intercellular and perivascular fluid accumulation that appeared to be reversible. In a study by Yang et al [30], guinea pigs were treated with irrradiation doses of 1 Gy for 30 days, and the inner ear structures were evaluated by histopathology. There was degeneration in the support cells, OHC of the organ of Corti, stria vascularis, vessel endothelial cells, reduced number of capillaries, and tissue atrophy. In the present study, high dose RT of 60
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Fig. 1. Histopathological images of the Control group: (a) normal histopathological appearance of the stria vascularis in the cochlea (arrow-head) (H&E x 400); (b) normal histopathological appearance of organ of Corti (outer hair cells and inner hair cells) in the cochlea (arrow-head) (H&E x 400); (c) normal histopathological appearance of the spiral ganglion in the cochlea (H&E x 200).
Fig. 2. Histopathological images of the radiotherapy (RT) group: (a) severe radiation damage of stria vascularis in the cochlea (arrow-head) (H&E x 400); (b) severe radiation damage of the organ of Corti in the cochlea (arrow-head) (H&E x 400); (c) severe radiation damage of the spiral ganglion in the cochlea (arrow) (H&E x 400).
Gy caused extensive damage to the inner ear, including the stria vascularis, outer hair cells, inner hair cells, and spiral ganglion cells.
In order to protect the cochlea from ototoxicity, numerous drugs have been used (eg, cisplatin, aminoglycosides, antioxidant agents such as Lmethionine, -lipoic acid, vitamin E, vitamin B, pantothenic acid, coenzyme A, piracetam, and
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Fig. 3. Histopathological images of the radiotherapy plus Lcarnitine (LC+RT) group: (a) mild radiation damage of stria vascularis in the cochlea (arrow) (H&E x 400); (b) mild radiation damage of the organ of Corti in the cochlea (arrow) (H&E x 400); (c) mild radiation damage of the spiral ganglion in the cochlea (head arrows) (H&E x 400). The radiation damage was less severe in the LC+RT group than in the RT group (see Fig. 2a,b,c)
aminoguanidine) [8-16]. L-carnitine (LC) is an antioxidant molecule with ubiquitous tissue distribution and cardioprotective, neuroprotective, and immunostimulant properties [31]. Studies have
shown that LC administration ameliorates postischemic reperfusion-induced oxidative damage of the kidney [32], retina [14], spinal-cord [33], and heart [34]. Studies in vivo and in vitro suggest that LC protects the myocardium against adriamycininduced cardiotoxicity [35,36] and the kidney and small intestine against cisplatin-induced injury [37] without interfering with its antitumor activity. Carnitine acts as an anti-apoptotic mediator [38,39]. Exposure of mouse spleen to low-frequency high-intensity magnetic field (MF) decreases splenocyte viability, leukocyte count, and mitogeninduced lymphocyte proliferation; LC ameliorates most of these adverse effects of MF, suggesting a possible immunoprotective role of LC [40]. Acetylcarnitine can increase the metabolic rate of mitochondria and improve mitochondrial oxygen utilization, which may counteract some toxic effects in ischemically injured tissue [41]. L-propionylcarnitine and LC act as superoxide scavengers, inhibit peroxidation of linoleic acid, and protect DNA from cleavage induced by H2O2 UV-photolysis [42,43]. LC may acts as a chelator to decrease the concentration of cytosolic iron, which may play an important role in free radical chemistry [44]. Acetyl-L-carnitine has a capacity to enhance the activities of non-enzymatic antioxidants, such as vitamin E and ascorbic acid [45], and to modulate the secretion of melatonin [46,47]. These are some of the reasons why we tested LC as a radioprotector in the present study. We are not aware of any previous investigation of LC protection against inner ear damage. The present study in guinea pigs shows that fractionated total cranial irradiation of 60 Gy (RT group) damaged the stria vascularis, spiral ganglion cells, OHC, and IHC of cochleas, and that LC administration (RT+LC group) decreased the radiation-induced cochlear damage. Thus, LC played a significant neuroprotective role after fractionated total cranial irradiation of 60 Gy. The authors speculate that LC protection against inner ear damage may be mediated by improving mitochondrial oxygen utilization and scavenging free-radicals. In summary, ip administration of L-carnitine resulted in beneficial reduction of radiation-induced ototoxicity in guinea pigs.
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