Since the discovery of X-rays, radiotherapy (RT) has beenused widely in both primary and adjuvant treatment of cancerdiseases, but some side effects of RT have occurred in addi-tion to these curative effects. One of these side effects is oto-toxicity. Hearing loss may be severe and uncomfortableenough to disrupt the life quality of some patients.1—4) Inorder to minimize or prevent otologic side effects induced byRT and various other drugs, studies have continued to thepresent day. These include pantothenic acid, coenzyme A, D-methionine, histidine, brain derived neurotrophic factorwith D-methionine, L-methionine, diethyldithiocarbamate, 4-methylthiobenzoic acid, ebselen, alpha lipoic acid, alpha to-copherol, vitamins B, A, C, E and K, zinc–copper-superoxidedismutase, nicotinamide, amino acids, choline, ATP, glu-curonic acid, chondroitin sulfate, corticoids, sulfhydryl com-pounds (i.e. panthotenic acid, glutathione, sodium thiosul-fate), carnitine and piracetam.5—15)

Piracetam (PIR) is a drug, with a fairly wide effect spec-trum. Also, it has been used in the treatment of epilepsy,cerebro-vascular occlusion, motor aphasy, amnesia and sud-den hearing loss, and good results have been obtained fromthe treatment.16—19) Piracetam (2-oxo-1-pyrrolidine-aceta-mine) is a low molecular weight derivative of gamma-aminobutyric acid. Different but complementary effects havebeen recognized, such as effects on cognitive function,platelet anti-aggregant, rheological and antioxidant mecha-nisms.20—22) Nootropic (cognitive) effects were the first estab-lished. These relate both to the ability to cross the blood–brain barrier and local vasomotor and metabolic effects, suchas increase in oxygen and glucose utilisation, decrease in thelactate/pyruvate ratio and restoration of regional metabolism

via the ATP pathway.21,22) Platelet anti-aggregant effectsmight be secondary to a direct inhibitory effect of piracetamon tromboxane A2 or tromboxane A2 synthetase in the chainof synthesis of platelet prostaglandins. The rheological ef-fects of piracetam, especially those related to red cells, areprobably related partly to cell membrane changes, such aslipid structure, prostaglandin synthesis, and degree of phos-phorylation of spectrin, especially in erythrocytes but also inwhite cells and platelets, and partly to qualitative changes insome plasma protein fractions such as fibrinogen, macro-globulins and von Willebrand’s factor.20)

The purpose of this study was to examine the protectiveeffects of parenteral piracetam supplementation on cochleardamage occurring in guinea pigs exposed to total cranium ir-radiation.

MATERIALS AND METHODS

Animals, Drugs and Irradiation All procedures includ-ing albino guinea pigs were carried out adhering to principlesof the Use of Animals in Research. The guinea pigs werequarantined for at least 1 d before irradiation, housed incages in a windowless laboratory room with automatic tem-perature (22�1 °C) and lighting controls (12 h light/12 hdark), and fed standard laboratory chow and water adlibitum.

Male albino guinea pigs (450—600 g body weight) (n�76) were randomly divided into three groups. Group 1 (Con-trol group) (n�11) received neither PIR nor irradiation, butreceived saline solution (3.5 ml/kg) i.p. and received sham ir-radiation for five successive days. Group 2 (RT group)

1460 Vol. 29, No. 7Biol. Pharm. Bull. 29(7) 1460—1465 (2006)

Effects of Piracetam Supplementation on Cochlear Damage Occuring inGuinea Pigs Exposed to Irradiation

Enver ALTAS,*,a Mustafa Vecdi ERTEKIN,b Ozan KUDUBAN,a Cemal GUNDOGDU,c Elif DEMIRCI,c andYavuz SUTBEYAZ

a

a Department of Otolaryngology, School of Medicine, Ataturk University; b Radiation Oncology, School of Medicine,Ataturk University; and c Pathology, School of Medicine, Ataturk University; 25240, Erzurum, Turkey.Received September 28, 2005; accepted February 13, 2006

In this study we aimed to determine the role of piracetam (PIR) in preventing radiation induced cochleardamage after total-cranium irradiation (radiotherapy; RT). Male albino guinea pigs used in the study were ran-domly divided into three groups. Group 1 (Control group) (n�11) received neither PIR nor irradiation, but re-ceived saline solution intraperitoneally (i.p.) and received sham irradiation. Group 2 (RT group) (n�32) was ex-posed to total cranium irradiation of 33 Gy in 5 fractions of 6.6 Gy/d for five successive days, with a calculated(aa/bb�3.5) biological effective dose of fractionated irradiation equal to 60 Gy conventional fractionation, then re-ceived saline solution for five successive days i.p. Group 3 (PIR�RT group) (n�33) received total cranium irradi-ation, plus 350 mg/kg per day PIR for five successive days i.p. After the last dose of RT, the guinea pigs were allsacrificed at the 4th, 24th and 96th hours, respectively. Their cochleas were enucleated for histopathologic exami-nation. It was observed that total cranium irradiation (RT group) promoted degeneration in stria vascularis(SV), spiral ganglion cells (SG), outer hair cells (OHC) and inner hair cell (IHC) of cochleas at these times(p�0.05). While in the PIR�RT group, there was no statistically significant difference on radiation-inducedcochlear degeneration in SV and OHC at 4th (p�0.05) and IHC at 4th, 24th hours (p�0.05), there was a signifi-cant difference on radiation-induced cochlear degeneration in SV and OHC at 24th and 96th hours (p�0.05),IHC at 96th hour (p�0.05) and SG at 4th, 24th and 96th hours (p�0.05). There was no any cochlear degenera-tion in the control group. Piracetam might reduce radiation-induced cochlear damage in the guinea pig. Theseresults are pioneer to studies that will be performed with PIR for radiation toxicity protection.

Key words piracetam; cochlear damage; gamma-irradiation

© 2006 Pharmaceutical Society of Japan∗ To whom correspondence should be addressed. e-mail: enveraltas@hotmail.com

(n�32) was exposed to total cranium irradiation of 33 Gy in5 fractions of 6.6 Gy/d for five successive days, with a calcu-lated (a /b�3.5) biological effective dose of fractionated irra-diation equal to 60 Gy conventional fractionation, plus re-ceiving saline solution (3.5 ml/kg) i.p. for five successivedays as a placebo. Group 3 (PIR�RT group) (n�33) re-ceived total cranium irradiation, plus 350 mg/kg per day PIR(UCB Pharma, Istanbul, Turkey) for five successive days i.p.In our study, 350 mg/kg/d dose of piracetam (i.e. 3.5 ml/kg)was used 1 h before the total cranium irradiation.

Prior to total cranium radiotherapy, the guinea pigs wereanesthetized with 50 mg/kg ketamin HCl (Pfizer, Istanbul)and placed on a plexiglas tray in a prone position. The guineapigs in the RT and PIR�RT groups were irradiated using acobalt-60 teletherapy unit (Picker, C-9, Maryland, NY,U.S.A.) from a source-to-surface distance of 80 cm, by7.5�7.5 cm anterior fields with 33 Gy to the total cranium in5 fractions of 6.6 Gy/d for five successive days; the calcu-lated (a /b�3.5) biological effective dose of fractionated irra-diation was equal to 60 Gy conventional fractionation. Thedose was calculated for the central axis at a depth of 1.5 cm.The dose rate was 0.65 Gy/min.

After the last dose of irradiation and/or drug, the guineapigs were anesthetized by i.p. administration of 100 mg/kg ofketamine hydrochloride (Pfizer) and 3 mg/kg of diazepam(Deva, Istanbul), and were sacrificed with overdoses(100 mg/kg) of pentobarbital (Abbott, Istanbul) this was ad-ministered to all of the control group (n�11), 12 and 11 ani-mals at the 4th hour from 2 and 3 groups, 10 and 12 animalsat the 24th hour, and 10 and 10 animals at the 96th hour, re-spectively. Their right and left temporal bones were removedwithin 5 min of sacrifice and it was reached to the tympanicbulla after removal of the lateral wall of the mastoid likeprocess of these temporal bones. Their left cochleas wereenucleated for histopathologic examination, and their righttemporal bones were used for another study.

Histopathological Evaluation The left tympanic bullaswere fixed in 10% neutral buffered formaldehyde solutionand kept in this solution at �4 °C for 24 h. For decalcifica-tion, specimens were stored in 10% ethylenediaminete-traacetic acid solution at 4 °C for 20 d. All specimens weredehydrated, embedded in paraffin and serially cut into 5 mmslices. Sections were stained with haematoxylin and eosin forlight microscopic examination by two independent patholo-gists. Changes in the organ of Corti (hydropic and vacuolardegeneration and loss of outer hair cells (OHC) and innerhair cell (IHC)); in spiral ganglion (SG) (cytoplasmic andnuclear condensation, nucleus and neuron loss); and in thestria vascularis (SV) (edema, vacuolization and loss of cells)were noted. While changes in SV were scored as absent (0),mild (1), moderate (2) or severe (3), the percentage of degen-erated cells in SG and the number of degenerated cells in theorgan of Corti were evaluated.

Statistical Analysis After necessary data had been col-lected, statistical analyses were made using the SPSS 11.0packet programme (Statistical Package for Social Science;Windows version 11.0). The results were given as means�standard deviation. Statistical significance of differences be-tween the groups was tested with the One-way ANOVA test.p�0.05 was accepted as statistically significant.

RESULTS

There was no cochlear degeneration in the control group(Table 1, Figs. 1, 4, 7). When the RT and PIR�RT groups

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Fig. 1. Stria Vascularis by Light Microscopy in the Cochleas of the Con-trol Group (H&E�400)

Fig. 2. Changes in Stria Vascularis (Severe Degeneration) by Light Mi-croscopy in the Cochleas of the Radiotherapy (RT) Group (H&E�200)

Fig. 3. Changes in Stria Vascularis (Mild Degeneration) by Light Mi-croscopy in the Cochleas of the Radiotherapy Plus Piracetam (PIR�RT)Group (H&E�200)

were compared with control group, cochlear damage in theguinea pigs at 4th, 24th and 96th hours was significant(p�0.05). We found that the total cranium irradiation (RT

group) promoted degeneration in the SV (severe hydropicand vacuolar degeneration) (Fig. 2), OHC and IHC (hy-dropic, vacuolar degeneration, and loss of cells) (Fig. 5) and

1462 Vol. 29, No. 7

Fig. 4. Organ of Corti (Outer Hair Cells and Inner Hair Cell) by Light Mi-croscopy in the Cochleas of the Control Group (H&E�400)

Fig. 5. Changes in Organ of Corti (Severe Degeneration, and Loss of Cellin Outer Hair Cells and Inner Hair Cell) by Light Microscopy in theCochleas of the Radiotherapy (RT) Group (H&E�200)

Fig. 6. Changes in Organ of Corti (Mild Degeneration in Outer Hair Cellsand Inner Hair Cell) by Light Microscopy in the Cochleas of the Radiother-apy Plus Piracetam (PIR�RT) Group (H&E�200)

Fig. 7. Spiral Ganglion by Light Microscopy in the Cochleas of the Con-trol Group (H&E�200)

Fig. 8. Changes in Spiral Ganglion (Inconspicuous Cytoplasmic and Nu-clear Condensation, Nucleus and Neuron Loss) by Light Microscopy in theCochleas of the Radiotherapy (RT) Group (H&E�400)

Fig. 9. Changes in Spiral ganglion (Mild Cytoplasmic and Nuclear Con-densation, and Loss of a Few Nuclei and Neuron) by Light Microscopy in theCochleas of the Radiotherapy Plus Piracetam (PIR�RT) Group (H&E�400)

SG (inconspicuous cytoplasmic and nuclear condensation,nucleus and neuron loss) (Fig. 8) of cochleas at 4th, 24th and96th hours (p�0.05) (Table 1). PIR supplementation(PIR�RT group) decreased damage in SV (mild hydropicand vacuolar degeneration) at 24th and 96th hours (Fig. 3),OHC (hydropic degeneration) at 24th and 96th hours andIHC (hydropic degeneration) (Fig. 6) and SG (cytoplasmicand nuclear condensation, and loss of a few nucleus and neu-ron) at 4th, 24th and 96th hours (Fig. 9), at 96th hour(p�0.05) the radiation-induced cochlear degeneration. But,none of these changes was not observed in SV and OHC at4th and in the IHC at 4th and 24th hours (p�0.05) (Tables 1,2).

DISCUSSION

The goal of radiation treatment is to deliver completelymeasured doses of ionizing radiation to a defined tumor vol-ume with the minimum acceptable injurious effects of ioniz-ing radiation to surrounding healthy tissue by eliminating

tumor cells, providing a high quality of life and prolongationof survival at reasonable cost to cancer patients.24) Cochleartoxicity may occur, however, during radiation therapy of headand neck cancer.1)

In the literature we found that cochlear damage increasedin proportion to RT dose. Kim and Shin25) applied a singledose of 2, 6, 10, or 15 Gy to guinea pigs, and evaluatedcochleas histopathologically. They found that OHC damageappeared with irradiation of more than 10 Gy, and IHC dam-age with irradiation of more than 15 Gy. They also found thatthe morphologic changes in SV were intercellular andperivascular fluid accumulation that have appeared to be a re-versible process. Young et al.26) carried out experimentalstudies in which they gave 2 Gy-dose ionized radiation toguinea pigs for 30 d, then evaluated the inner ear structuremicroscopically. They detected degeneration in support cellsand in OHC of the Corti organ, in vessel endoteliocytes, re-duction in the number of capillaries, atrophy and degenera-tion in SV. In the present study at the applied high RT dose of60 Gy, we demonstrated that irradiation caused damage in all

July 2006 1463

Table 1. Scoring of Changes in Organ of Corti (Hydropic and Vacuolar Degeneration and Loss of Outer Hair Cells and Inner Hair Cell); in Spiral Ganglion(Cytoplasmic and Nuclear Condensation, Nucleus and Neuron Loss); and in Stria Vascularis (Hydropic and Vacuolar Degeneration) by Light Microscopy inCochleas of Control, Radiotherapy (RT) and RT Plus PIR (PIR�RT) Groups at 4th, 24th and 96th Hours

ControlRT RT RT PIR�RT PIR�RT PIR�RT

4th h 24th h 96th h 4th h 24th h 96th h(n�11) (n�12) (n�10) (n�10) (n�11) (n�12) (n�10)

Degeneration in SV0 11 0 0 0 0 0 41 0 0 0 0 0 5 42 0 7 4 2 11 7 23 0 5 6 8 0 0 0

N of deg cells in SG 0 66.50 80.80 88.80 53.09 42.50 31.00(% mean�S.D.) �4.27 �3.15 �3.01 �3.01 �2.84 �3.16

N of deg cells in OHC0 11 0 0 0 0 0 41 0 0 0 0 4 5 32 0 7 4 2 4 5 23 0 5 6 8 3 2 1

N of deg cells in IHC0 11 7 4 3 9 11 101 0 5 6 7 2 1 0

RT: radiotherapy groups; RT�PIR: radiotherapy plus piracetam groups; h: hour; n: number; Deg: degenerated; SV: stria vascularis; SG: spiral ganglion; OHC: outer hair cells;IHC: inner hair cell. Changes in SV were scored as none (0), mild (1), moderate (2) or severe (3).

Table 2. Statistically Analysis of Changes in Organ of Corti (Hydropic and Vacuolar Degeneration and Loss of Outer Hair Cells and Inner Hair Cell); inSpiral Ganglion (Cytoplasmic and Nuclear Condensation, Nucleus and Neuron Loss); and in Stria Vascularis (Hydropic and Vacuolar Degeneration) by LightMicroscopy in Cochleas of Control, Radiotherapy (RT) and RT Plus PIR (PIR�RT) Groups at 4th, 24th and 96th Hours

ControlRT RT RT PIR�RT PIR�RT PIR�RT

4th h 24th h 96th h 4th h 24th h 96th h(n�11) (n�12) (n�10) (n�10) (n�11) (n�12) (n�10)

Degeneration 0� 3.42� 3.60� 3.80� 3.00� 2.58� 1.80�in SV 0.00b,c,d,e,f,g 0.51a,e,f,g 0.51a,e,f,g 0.42a,e,f,g 0.00a,c,d,f,g 0.51a,b,c,d,g 0.76a,b,c,e,f

N of deg cells 0� 66.50� 80.80� 88.80� 53.09� 42.50� 31.00�in SG 0.00b,c,d,e,f,g 4.27a,c,d,e,f,g 3.15a,b,d,e,f,g 3.01a,b,c,e,f,g 3.01a,b,c,d,f,g 2.84a,b,c,d,e,g 3.16a,b,c,d,e,f

N of deg cells 0� 3.42� 3.60� 3.80� 2.91� 2.75� 2.00�in OHC 0.00b,c,d,e,f,g 0.51a,f,g 0.51a,e,f,g 0.42a,e,f,g 0.83a,c,d,g 0.75a,b,b,d,g 1.05a,b,c,d,e

N of deg cells 0� 1.42� 1.60� 1.70� 1.18� 1.08� 1.00�in IHC 0.00b,c,d,e,f,g 0.51a,g 0.51a,e,f,g 0.48a,e,f,g 0.40a,c,d 0.28a,c,d 0.00a,b,c,d

RT: radiotherapy groups; PIR�RT: radiotherapy plus piracetam groups; h: hour; n: number; Deg: degenerated; SV: stria vascularis; SG: spiral ganglion; OHC: outer hair cells;IHC: inner hair cell. a p�0.05 vs. RT 4th h group, b p�0.05 vs. RT 4th h group, c p�0.05 vs. RT 24th h group, d p�0.05 vs. RT 96th h group, e p�0.05 vs. RT�PIR 4th h group, f p�0.05 vs. RT�PIR 24th h group, g p�0.05 vs. RT�PIR 96th h group.

the investigated parameters of SV, OHC, IHC and SG.Oxidative stress has an important place in ototoxicity

pathophysiology.27) Previous studies have suggested that cis-platin or aminoglycoside ototoxicity was related to an in-crease in lipid peroxidation and a decrease in endogenous an-tioxidant enzyme status.28,29) In cisplatin and aminoglyco-side-induced ototoxicity, it has been reported that L-methion-ine, alpha lipoic acid, vitamin E, vitamin B, pantothenic acid,co-enzyme A, and piracetam were used as protective agentsbecause of their antioxidant properties.8—15)

Exposure of cells to ionizing radiation cause oxidativestress because it occurs through which a variety of reactiveoxygen species, such as the superoxide radical (O2

·�), thehydrogen peroxide (H2O2) and the hydroxyl radical (OH�)that are associated with radiation-induced cytotoxicity30) Al-though all respiring cells are equipped with protective en-zymes such as SOD and CAT or GSH-Px, increased oxida-tive stress in cells stemming from ionizing radiation mayoverwhelm the protective systems, leading to cell injury.31)

Consequently, piracetam is a drug that has antioxidant,22,32,33)

platelet antiaggregant, rheological,20—22) anti-inflammatory,34)

antiapoptotic, cytoprotective35) and immunomodulatory ef-fects.36,37) Minkova et al.38) reported that 200 mg/kg pirac-etam combined with vitamin E 20 mg/kg and anthocyans50 mg/kg has a radioprotective effect. They found that thecombination significantly increased survival rate (50%) inmice, blood-forming organs underwent less radiation dam-age, and recovery processes in them were stimulated. In a re-cent study, we demonstrated that piracetam prevented ototox-icity by interfering with the neuroprotective mechanism inthe guinea pigs in vivo. We also determined that piracetamimproved the ototoxic effects of combined cisplatin–genta-micin upon auditory pathways from cochlea to middle brain.Thereby, we deduced that, because of the protective effects ofpiracetam, it was able to provide more effective treatment ofotorhinolaryngologic, neurologic, infectious and oncologicdiseases by reducing combined cisplatin–gentamicin ototoxi-city.15) In terms of the above expressed information, one canallege that piracetem might play a role by increasing oxy-genation in the tumor cells with its rheological effects anddecreasing apoptosis in surrounding healthy cells with its an-tiapoptotic effects in the prevention of radiation-induced oxidative damage. Animal studies showed that rats treatedchronically with 100 to 1000 mg/kg orally for 6 months didnot show any toxic effect; Nor were any teratogenic effectsfound.39) Koskiniemi et al.40) reported that a dose of 24 g/dfor two weeks is highly beneficial, more effective than lowerdoses as an effective and safe medication in patients. Thus, apiracetam dose of 350 mg/kg/d in our study was similar to24 g/d used in a 70 kg of human. The plasma eliminationhalf-life was short in all the species studied, ranging from amean of 1.8—2.0 and 4.5 h following administration in ratsand man, respectively.41,42) Piracetam was used for 1 h to pre-vent barbiturate intoxication in rabbit.13) We also used it 1 hbefore the total cranium irradiation. These are some of otherreasons why we used piracetam as a potent radioprotectoragent in the present study.

We found in this study that fractionated total cranium irra-diation of 60 Gy (RT group) promoted damage in SV, SG,OHC and IHC of cochleas, and piracetam supplementation(PIR�RT group) decreased the radiation-induced cochlear

damage.In conclusion, piracetam seems to have beneficial effects

in the reduction of radiation-induced ototoxicity. These arepioneer results for studies that will be performed with PIR toprotect from radiation toxicity. It would be worthwhile study-ing the effects of PIR supplements in radiation-treated cancerpatients, in the hope of reducing radiation-induced toxicity.

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