Role of N-acetyl cysteine and acetyl-l-carnitine combination treatment on DNA-damage-related genes induced by radiation in HEI-OC1 cells.

PURPOSE: The aim of the present study was to evaluate the effect of acetyl-l-carnitine (ALC) and N-acetyl cysteine (NAC) on ionizing radiation (IR)-induced cytotoxicity and change in DNA damage-related genes in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. METHODS: HEI-OC1 cells were irradiated with 5 Gy radiation and treated by eight combinations of NAC and/or ALC: control, NAC, ALC, IR, NAC + IR, ALC + NAC, ALC + IR, and ALC + NAC + IR. Cell viability, apoptotic cell death, and DNA damage were measured at the 72nd hour. Eighty-four IR-induced DNA-damage-related genes were determined by RT-PCR gene array and >10-fold changes were considered significant. RESULTS: IR decreased cell viability by about 50% at 72 hours of incubation. In particular, the ALC and/or NAC combination before IR protected the HEI-OC1 cells (p < .05). Single and combination treatment prior to IR led to lower apoptotic cell death (p < .05). There was a significant lower DNA damage in ALC + NAC + IR group compared to IR group (p < .05). Expressions of Brca2, Xpc, Mlh3, Rad51, Xrcc2, Hus1, Rad9a, Cdkn1a, Gadd45a which are the DNA-repair genes were found to be significantly higher in NAC + ALC + IR group than those in individual treatment of ALC or NAC. CONCLUSIONS: ALC and/or NAC treatment prior to IR led to higher cell viability and lower apoptotic cell damage compared to the IR group. The results of the study show that the ALC + NAC combination treatment inhibits DNA damage and induces DNA-repair genes to repair radiation damage, and this combination treatment is more effective against radiation-induced DNA damage than NAC or ALC therapy individually.

Id1/NR2B Receptor Pathway Regulates Rat Cochlear Sensory Epithelial Cell Survival after Radiation.

Survival of cochlear sensory epithelial cells may be regulated by inhibitor of differentiation-1 (Id1) and the N-methyl-D-aspartic acid (NMDA) receptor. However, it is unclear whether Id1 and the NMDA receptor are involved in the radiation-mediated survival of rat cochlear sensory epithelial cells. Here, we show that the percentage of apoptotic cells increased, the percentage of cells in the S phase decreased, Id1 mRNA and protein expression decreased and the NMDA receptor subtype 2B (NR2B) mRNA and protein level increased in OC1 cells after radiation. Cells infected with the Id1 gene exhibited higher Id1 mRNA and protein levels and lower NR2B mRNA and protein levels than the control cells. In contrast, after transfection of the Id1 siRNA into OC1 cells, Id1 mRNA and protein expression decreased and NR2B mRNA and protein expression increased relative to that of the control group. Additionally, treatment with ifenprodil for 24 h before radiation reduced apoptosis and increased the percentage of cells in the S phase. Our results suggest that Id1 and NR2B might regulate the survival of OC1 cells following radiation.

Continuous exposure to low-frequency noise and carbon disulfide: Combined effects on hearing.

Carbon disulfide (CS2) is used in industry; it has been shown to have neurotoxic effects, causing central and distal axonopathies.However, it is not considered cochleotoxic as it does not affect hair cells in the organ of Corti, and the only auditory effects reported in the literature were confined to the low-frequency region. No reports on the effects of combined exposure to low-frequency noise and CS2 have been published to date. This article focuses on the effects on rat hearing of combined exposure to noise with increasing concentrations of CS2 (0, 63,250, and 500ppm, 6h per day, 5 days per week, for 4 weeks). The noise used was a low-frequency noise ranging from 0.5 to 2kHz at an intensity of 106dB SPL. Auditory function was tested using distortion product oto-acoustic emissions, which mainly reflects the cochlear performances. Exposure to noise alone caused an auditory deficit in a frequency area ranging from 3.6 to 6 kHz. The damaged area was approximately one octave (6kHz) above the highest frequency of the exposure noise (2.8kHz); it was a little wider than expected based on the noise spectrum.Consequently, since maximum hearing sensitivity is located around 8kHz in rats, low-frequency noise exposure can affect the cochlear regions detecting mid-range frequencies. Co-exposure to CS2 (250-ppm and over) and noise increased the extent of the damaged frequency window since a significant auditory deficit was measured at 9.6kHz in these conditions.Moreover, the significance at 9.6kHz increased with the solvent concentrations. Histological data showed that neither hair cells nor ganglion cells were damaged by CS2. This discrepancy between functional and histological data is discussed. Like most aromatic solvents, carbon disulfide should be considered as a key parameter in hearing conservation régulations.

Dexamethasone Protects Against Radiation-induced Loss of Auditory Hair Cells In Vitro.

HYPOTHESIS: Dexamethasone (DXM) protects against radiation-induced loss of auditory hair cells (HCs) in rat organ of Corti (OC) explants by reducing levels of oxidative stress and apoptosis. BACKGROUND: Radiation-induced sensorineural hearing loss (HL) is progressive, dose-dependent, and irreversible. Currently, there are no preventative therapeutic modalities for radiation-induced HL. DXM is a synthetic steroid that can potentially target many of the pathways involved in radiation-induced ototoxicity. METHODS: Whole OC explants were dissected from 3-day-old rat cochleae exposed to specific dosages of single-fraction radiation (0, 2, 5, 10, or 20 Gy), were either untreated or treated with DXM (75, 150, 300 µg/mL), and then cultured for 48 or 96 hours. Confocal microscopy for oxidative stress (CellRox, 48 h) and apoptosis (TUNEL assay, 96 h) and fluorescent microscopy for viable HC counts (fluorescein isothiocyanate-phalloidin, 96 h) were performed. Analysis of variance and Tukey post hoc testing were used for statistical analysis. RESULTS: Radiation exposure initiated dose-dependent losses of inner and outer HCs, predominantly in the basal turns of the OC explants. DXM protected against radiation-induced HC losses in a dose-dependent manner. DXM significantly reduced levels of oxidative stress and apoptosis in radiation-injured OC explants (p < 0.001). CONCLUSIONS: Radiation-initiated HC losses were dose-dependent in OC explants. DXM treatment protected explant HCs against radiation-initiated losses by decreasing the levels of oxidative stress and apoptosis. DXM may potentially be a therapeutic modality for preventing radiation-induced HL; further in vivo studies are necessary.

Inhibition of p38 mitogen-activated protein kinase ameliorates radiation-induced ototoxicity in zebrafish and cochlea-derived cell lines.

Radiation is a widely used treatment for head and neck cancers, and one of its most severe side effects is ototoxicity. Radiation-induced ototoxicity has been demonstrated to be linked to the increased production of ROS and MAPK. We intended to investigate the effect of p38 inhibition on radiation-induced ototoxicity in cochlea-derived HEI-OC1 cells and in a zebrafish model. The otoprotective effect of p38 inhibition against radiation was tested in vitro in the organ of Corti-derived cell line, HEI-OC1, and in vivo in a zebrafish model. Radiation-induced apoptosis, mitochondrial dysfunction, and an increase of intracellular NO generation were demonstrated in HEI-OC1 cells. The p38-specific inhibitor, SB203580, ameliorated radiation-induced apoptosis and mitochondrial injury in HEI-OC1 cells. p38 inhibition reduced radiation-induced activation of JNK, p38, cytochrome c, and cleavage of caspase-3 and PARP in HEI-OC1 cells. Scanning electron micrography showed that SB203580 prevented radiation-induced destruction of kinocilium and stereocilia in zebrafish neuromasts. The results of this study suggest that p38 plays an important role in mediating radiation-induced ototoxicity and inhibition of p38 could be a plausible option for preventing radiation ototoxicity.

Analysis of the cytoprotective effect of amifostine on the irradiated inner ear of guinea pigs: an experimental study.

UNLABELLED: Radiation can cause damage to the inner ear, from a simple hearing loss all the way to profound deafness. Amifostine is a cytoprotective substance extensively used during radio-chemotherapy for malignant tumors. AIM: the objective of the present investigation was to establish the antioxidant and radioprotective effects of amifostine on the organ of Corti of albino guinea pigs irradiated in the head and neck region. MATERIALS AND METHODS: An experimental study conducted on four groups of guinea pigs were used; One group received only amifostine, one group was submitted to a single dose of 350 cGy and the other two were similarly irradiated but received amifostine doses of 100 or 200 mg/kg. All animals were slaughtered 30 days after the experiment, their bullae were removed and the damaged outer hair cells were counted. RESULT: The extent of injury was lower in the outer hair cells of the two groups treated with amifostine compared to the group that was only irradiated. There was no difference between the group treated with 100 and 200 mg/kg of amifostine. The group that received only amifostine had no cochlear damage. CONCLUSION: Amifostine is an effective cytoprotective substance in the Organ of Corti of irradiated guinea pigs.

Analysis of the cytoprotective effect of amifostine on the irradiated inner ear of guinea pigs: an experimental study / Análise do efeito citoprotetor da amifostina na orelha interna irradiada de cobaias: estudo experimental

Radiation can cause damage to the inner ear, from a simple hearing loss all the way to profound deafness. Amifostine is a cytoprotective substance extensively used during radio-chemotherapy for malignant tumors. AIM: the objective of the present investigation was to establish the antioxidant and radioprotective effects of amifostine on the organ of Corti of albino guinea pigs irradiated in the head and neck region. MATERIALS AND METHODS: An experimental study conducted on four groups of guinea pigs were used; One group received only amifostine, one group was submitted to a single dose of 350 cGy and the other two were similarly irradiated but received amifostine doses of 100 or 200 mg/kg. All animals were slaughtered 30 days after the experiment, their bullae were removed and the damaged outer hair cells were counted. RESULT: The extent of injury was lower in the outer hair cells of the two groups treated with amifostine compared to the group that was only irradiated. There was no difference between the group treated with 100 and 200 mg/kg of amifostine. The group that received only amifostine had no cochlear damage. CONCLUSION: Amifostine is an effective cytoprotective substance in the Organ of Corti of irradiated guinea pigs.

A radiação pode causar lesão na orelha interna podendo provocar surdez sensório-neural e inclusive levar à anacusia. A amifostina é uma substância citoprotetora seletiva de tecidos sadios, amplamente utilizada durante a radio e quimioterapia de tumores malignos. OBJETIVO: O objetivo deste estudo experimental foi verificar se existe efeito antioxidante e radioprotetor da amifostina no órgão de Corti de cobaias albinas irradiadas em região de cabeça e pescoço. MATERIAL E MÉTODO: O estudo realizado envolveu quatro grupos de animais: um grupo foi submetido à irradiação em dose única de 350cGy. Dois grupos receberam a mesma dose de radiação, porém receberam doses de 100 e 200mg/kg de amifostina, 30 minutos antes da irradiação. Um grupo recebeu apenas amifostina, na dose de 200mg/Kg. Todas as cobaias foram sacrificadas 30 dias após o experimento e suas bulas retiradas para estudo em microscópio de varredura. RESULTADO: O grau de lesão das células ciliadas externas foi menor nos dois grupos que receberam a amifostina que no grupo apenas irradiado. Não foi encontrada diferença de proteção entre os grupos que receberam doses de 100 e 200mg/kg de amifostina. Não houve lesão no grupo que recebeu apenas amifostina. CONCLUSÃO: Amifostina mostrou ser um radioprotetor do órgão de Corti de cobaias albinas irradiadas.

Effects of piracetam supplementation on cochlear damage occurring in guinea pigs exposed to irradiation.

In this study we aimed to determine the role of piracetam (PIR) in preventing radiation induced cochlear damage after total-cranium irradiation (radiotherapy; RT). Male albino guinea pigs used in the study were randomly divided into three groups. Group 1 (Control group) (n=11) received neither PIR nor irradiation, but received saline solution intraperitoneally (i.p.) and received sham irradiation. Group 2 (RT group) (n=32) was exposed to total cranium irradiation of 33 Gy in 5 fractions of 6.6 Gy/d for five successive days, with a calculated (alpha/beta=3.5) biological effective dose of fractionated irradiation equal to 60 Gy conventional fractionation, then received saline solution for five successive days i.p. Group 3 (PIR+RT group) (n=33) received total cranium irradiation, plus 350 mg/kg per day PIR for five successive days i.p. After the last dose of RT, the guinea pigs were all sacrificed at the 4th, 24th and 96th hours, respectively. Their cochleas were enucleated for histopathologic examination. 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-induced cochlear degeneration in SV and OHC at 4th (p>0.05) and IHC at 4th, 24th hours (p>0.05), there was a significant 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 degeneration in the control group. Piracetam might reduce radiation-induced cochlear damage in the guinea pig. These results are pioneer to studies that will be performed with PIR for radiation toxicity protection.

[Electromechanical transduction: influence of the outer hair cells on the motion of the organ of Corti]. / Elektromechanische Transduktion: Einfluss der äusseren Haarsinneszellen auf das Bewegungsmuster des Corti-Organs.

BACKGROUND: The somatic electromotility of the outer hair cells can be induced by an extracellular electrical field. This enables us to investigate the electromechanically induced motion of the organ of Corti. METHODS: The electrically induced motion of the guinea-pig organ of Corti was measured with a laser Doppler vibrometer in three cochlear turns at ten radial positions on the reticular lamina (RL) and six on each of the upper and lower surfaces of the tectorial membrane (TM). RESULTS AND CONCLUSIONS: We found a complex vibration pattern of the RL and TM, leading to a stimulus synchronous modulation of the depth of the subtectorial space in the region of the inner hair cells (IHCs). This modulation causes radial fluid motion inside the space up to at least 3 kHz. This motion is capable of deflecting the IHC stereocilia and provides an amplification mechanism additional to that associated with basilar-membrane motion.

Local mechanical stimulation of the hearing organ by laser irradiation.

Light produces force when interacting with matter. Such radiation pressure may be used to accelerate small objects along the beam path of a laser. Here, we demonstrate that a moderately powerful laser can deliver enough force to locally stimulate the hearing organ, in the absence of conventional sound. Damped mechanical oscillations are observed following brief laser pulses, implying that the organ of Corti is locally resonant. This new method will be helpful for probing the mechanical properties of the hearing organ, which have crucial importance for the ear's ability to detect sound.

Effects of 900 MHz electromagnetic fields exposure on cochlear cells' functionality in rats: evaluation of distortion product otoacoustic emissions.

In recent years, the widespread use of mobile phones has been accompanied by public debate about possible adverse consequences on human health. The auditory system is a major target of exposure to electromagnetic fields (EMF) emitted by cellular telephones; the aim of this study was the evaluation of possible effects of cellular phone-like emissions on the functionality of rat's cochlea. Distortion Products OtoAcoustic Emission (DPOAE) amplitude was selected as cochlea's outer hair cells (OHC) status indicator. A number of protocols, including different frequencies (the lower ones in rat's cochlea sensitivity spectrum), intensities and periods of exposure, were used; tests were carried out before, during and after the period of treatment. No significant variation due to exposure to microwaves has been evidenced.

Effects of exposure of the ear to GSM microwaves: in vivo and in vitro experimental studies.

The effects of mobile phone (GSM) microwaves on the ears of guinea pigs were investigated in two in vivo experiments and one in vitro experiment. In the first experiment, three groups of eight guinea pigs had their left ear exposed for 1 h/day, 5 days/week, for 2 months, to GSM microwaves (900 MHz. GSM modulated) at specific absorption rates (SARs) of 1, 2 and 4 W/kg respectively, and a fourth group was sham-exposed. Distortion-product otoacoustic emissions (DPOAEs) were measured for each ear before exposure, at the end of the 2-month exposure period, and 2 months later. In the second experiment, the same protocol was applied to eight sham-exposed and 16 exposed guinea pigs at 4W/kg, but the auditory brainstem response (ABR) thresholds were monitored. Repeated-measures ANOVA showed no difference in DPOAE amplitudes or in ABR thresholds between the exposed and non-exposed ears and between the sham-exposed and exposed groups In the course of the second experiment, acute effects were also investigated by measuring once, in all animals, ABR thresholds just before and just after the 1-h exposure: no statistically significant difference was observed. In vitro, the two organs of Corti (OCs) of newborn rats (n=15) were isolated and placed in culture. For each animal, one OC was exposed for 24-48 h to 1 W/kg GSM microwaves, and the other was sham-exposed. After 2-3 days of culture, all OCs were observed under light microscopy. They all appeared normal to naive observers at this stage of development. These results provided no evidence that microwave radiation, at the levels produced by mobile phones, caused damage to the inner ear or the auditory pathways in our experimental animals.

Influence of pulsed laser irradiation on the morphology and function of the guinea pig cochlea.

Recent experimental and clinical studies have demonstrated that several pulsed laser systems are also suitable for stapedotomy. The aim of the study was to investigate morphological and functional inner ear changes after irradiating the basal turn of the guinea pig cochlea with two pulsed laser systems of different wavelengths. The Er:YSGG (lambda=2.78 mcm) and Ho:YAG (lambda=2.1 mcm) lasers were used applying the laser energies necessary for perforating a human stapes footplate. The cochleas were removed 90 min, 1 day, 2 weeks, or 4 weeks after laser application. Acoustic evoked potentials (compound action potentials) were measured before and after laser application and at the above times immediately before removal of the cochleas. The organ of Corti was examined by scanning electron microscopy. Application of Er:YSGG laser parameters effective for stapedotomy had no adverse effects on Corti's organ in the guinea pig cochlea. On the other hand, effective Ho:YAG laser parameters cause damage to the outer hair cells with fusion of stereocilia and formation of giant cilia leading to partial or total cell loss. The inner hair cells and supporting cells were usually normal. These morphological data show a good correlation with the electrophysiological measurements. Our results clearly demonstrate that, besides achieving efficient bone management, the Er:YSGG laser has high application safety. On the other hand, the Ho:YAG laser is not well tolerated in our animal study. Its use in stapedotomy would be unreliable and dangerous for the inner ear.

The role of radiation in delayed hearing loss in nasopharyngeal carcinoma.

Although radiation damage to the inner ear can be induced experimentally in animals, its incidence in humans as a complication of therapy for nasopharyngeal carcinoma (NPC) remains a matter of conjecture due to inadequate histological validation. A case of advanced NPC with a mixed conductive and neurosensory hearing loss is presented that at necropsy showed tumour invasion of the VIIIth cranial nerve in the internal auditory meatus with associated infection. The architecture of the organ of Corti was well-preserved despite heavy doses of radiation but degeneration, the cause of which could not be substantiated, was present in the auditory nerve pathway. A diagnosis of radiation damage to the end-organ of hearing in the absence of supporting histological evidence should be made with caution.

[Response of spiral organ cells on laser radiation to the region of tympanic graft (experimental study]. / Reaktsiia kletok spiral'nogo organa na lazernoe vozdeistvie v oblasti timpanal'nogo loskuta (éksperimental'noe issledovanie).

Spiral organ cells ultrastructure was studied electron microscopically after exposure to high-energy NIAG-Nd-laser radiation in contactless impulse regimen (1.06 microns, 20-40 W, 100 microseconds) in experimental tympanoplasty. The above laser radiation did not entail gross destruction in receptor and pillar cells in the peripheral compartment of the organ of hearing. Irrespective of the radiation power 3 days after the exposure some irradiated receptor cells showed insignificant changes: mitochondrial vacuolization, altered cuticular plate. 7 days after the exposure a positive response was registered in the majority of the receptor cells and in some pillar cells. This reflects activization of intracelluler biosynthetic processes.

Laser scanning confocal microscopy of the hearing organ: fluorochrome-dependent cellular damage is seen after overexposure.

In order to combine laser confocal microscopy with physiological measurements, a number of conditions have to be met: the dye must not be toxic to the cells the laser light itself must not damage the cells; and the excitation of the fluorochrome during imaging must not generate products with toxic effects. We have investigated these conditions the hearing organ of the guinea pig. Two dyes were used, namely, calcein-AM, which is metabolized in vital cells to a fluorescent product in the cytoplasm, and a lipophilic membrane dye. The effect of the dyes on cell function was tested in the intact hearing organ, maintained in the isolated temporal bone, by measuring the electrophysiological potentials generated by the sensory cells in response to tone pulses. The loading of the cells with the dyes had no adverse effects. The effect of the laser beam was explored on isolated coils from the cochlea. In two preparations, the specimens viewed in the confocal system were fixed and processed for electron microscopy. Identified cells were followed before, during, and after laser exposure and could ultimately be examined at the ultrastructural level. Exposure to the laser beam did not cause damage in unstained cells, even at high intensities. In stained tissue, confocal microscopy could safely be performed at normal beam intensity without causing ultrastructural changes. At high intensities, about 100 times normal for 60 times as long, irradiation damage was seen that was selective in that the cells stained with the different dyes exhibited damage at the different sites corresponding to the subcellular location of the dyes. Cells stained with calcein showed lysis of mitochondria and loss of cytoplasmic matrix, whereas cells stained with the styryl membrane dye showed swelling of subsurface cisternae, contortion of the cell wall, and shrinkage. The styryl dyes, in particular, which selectively stain the sensory and neuronal cells in the organ of Corti, could be exploited for phototoxic use.

Replacement of hair cells after laser microbeam irradiation in cultured organs of corti from embryonic and neonatal mice.

This study examined the potential for hair cell regeneration in embryonic and neonatal mouse organs of Corti maintained in vitro. Small numbers of hair cells were killed by laser microbeam irradiation and the subsequent recovery processes were monitored by differential interference contrast (DIC) microscopy combined with continuous time-lapse video recordings. Replacement hair cells were observed to develop in lesion sites in embryonic cochleae and on rare occasions in neonatal cochleae. In embryonic cochleae, replacement hair cells did not arise through renewed proliferation, but instead developed from preexisting cells that changed from their normal developmental fates in response to the loss of adjacent hair cells. In cochleae established from neonates, lost hair cells usually were not replaced, but 11 apparently regenerated hair cells and a single hair cell labeled by 3H-thymidine were observed as rare responses to the creation of hair cell lesions in these organs. The results indicate that the organ of Corti can replace lost hair cells during embryonic and on rare occasions during early neonatal development. The ability of preexisting cells to change their developmental fates in response to hair cell death is consistent with the hypothesis that during embryonic development hair cells may inhibit neighboring cells from specializing as hair cells. In neonatal cultures, the rare occurrence of apparently regenerated hair cells indicates that some cells in the postembryonic organ of Corti retain response mechanisms that can lead to self-repair.

Interaural correlations in normal and traumatized cochleas: length and sensory cell loss.

Sizable intraspecies variations have been found in both the length of the organ of Corti (OC) and the amount of damage resulting from exposure to a particular ototraumatic agent. These variations have made it difficult to address certain research questions such as the susceptibility of the previously injured ear to further damage. If intra-animal correlation is high, the variability problem could be circumvented by using the two ears from a given animal for different aspects of the same study. Therefore, correlation coefficients were calculated for OC length and for percentage of missing inner (IHCs) and outer hair cells (OHCs) in a large sample of chinchillas which included controls and animals which had been exposed to noise or treated with ionizing radiation. The correlation coefficients were +0.96 for OC length, +0.93 for IHC loss, and +0.97 for OHC loss.

Dose rate to the inner ear during Mössbauer experiments.

The most widely used technique for studying vibrations of the inner ear utilises the Mössbauer effect; this requires placement of a radioactive source on the basilar membrane. This source, although small in size and less than 37 MBq (1 mCi) in strength, is placed in close proximity to sensitive receptor cells. Using a series solution for the radiation field of a rectangular source the absorbed dose rate delivered to receptor cells at various depths and at points off-axis from the centre of the source is calculated. It is concluded that the dose delivered during the course of a Mössbauer experiment may well be sufficient to damage receptor cells and cause a loss of response.