| IntroductionThe use of aminoglycosides antibiotics (AmAn) has declined significantly in recent years due to the development of new antibiotics with fewer side effects. Nevertheless, AmAn are still common treatment choices for enterococcal, mycobacterial and serious infections induced by gram-negative bacteria (Swan,1997; Edson and Terrell,1999). In 1980s, there were about 2 million US patients per year to that had received treatment of AmAn.At present most assessments of the clinical incidence of hearing loss only provide conservative estimations. Hearing loss begins at the higher audible frequencies (in humans,16-18 kHz), however routine clinical auditory testing can only achieve 8 kHz, which is then further complicated by interfering noise.Despite these limitations, studies showed that cochleotoxicity of most commonly used aminoglycosides may occur in up to about 20% of patients and vestibular balance may be affected at a rate of about 15%(Fee et al,1980; Moore et al,1984; Lerner et al,1986).The problem of ototoxicity is most prevalent in developing countries. Due to their high efficacy and low cost, AmAn are still the most common the drugs of choice worldwide. Some safety precautions such as monitoring of serum levels or auditory function are hardly available or at all in these countries. Therefore, the incidence of hearing loss is estimated to be significantly higher than the 20% in developed countries. Indeed, studies from southern China revealed that 2/3 of all deaf-mutism in that area was due to the administration of aminoglycosides to children.A worldwide resurgence of tuberculosis (Shafer et al,1989) is the other important reason for the frequent use of AmAn. The incidence of tuberculosis became increasing recently because of its airborne transmission. The WHO expects tuberculosis to be the most devastating killer disease of this decade. Since the spread of tuberculosis is related to the emergence of multiple-drug-resistant bacterial strains, it necessitates combination drug therapy of which aminoglycosides (primarily streptomycin and amikacin) are an integral part. Result of study showed that kanamycin-induced hearing loss in tuberculosis patients has been found hearing loss in 80% of the patients (Brouet et al, 1959).In summary, it is necessary to further investigate the mechanisms underlying AmAn ototoxicity in order to minimize and eventually prevent the ototoxicity side effects and therefore to protect the hearing function from damaging, and take full advantage of the low cost and valuable antibacterial effects of AmAn.At present, studies on AmAn ototoxicity indicate that drugs delivered via systemic administration which caused the damage of cochlear hair cells, spiral ganglion cells, and vestibular hair cells, and induced the cell degeneration, apoptosis, and loss. Eventually, sensory epithelium will be all replaced by non-specific pavement epithelium, which causes irreversible hearing loss. In most cases, the dosage and duration of treatment determine the extent and severity of ototoxic damage. Another major influence factor appeared to be nutritional and physiological state of patients receiving treatment. In addition, impaired kidney function may limit AmAn excretion and therefore lead to accumulation of drugs, increase the incidence of ototoxicity; Drug-drug interactions can also increase the risk of AmAn ototoxicity, For example, the combination of loop diuretic, acidum ethacrynicum, and AmAn will greatly increase AmAn ototoxicity. On the other hand, the fact that certain groups of people, such as people with inherited mutant genes, pregnant women and infants, are more susceptible to AmAn ototoxicity may provide some clues to prevention of AnAn ototoxicity.Pathological changes induced by AmAn ototoxicity in cochlear appeared in OHCs in basilar membrane at early, and lately in IHCs which are real transducer which transduce mechanical signals induced by sound stimuli into auditory signals in auditory afferent nerve. Thereby the changes of morphology and functions of OHCs are still the focus of studying of mechanisms of ototoxicity induced by AmAn. Zheng J and colleagues (2000) found a novel motor protein-Prestin, which is very different to other motor proteins, for example, myosin, kinesin, dynein and so on, independent on ATP and Ca2+; which changes protein conformation in voltage-dependent directly, promptly, reversibly, causing OHCs somatic mechanical motion in response to changes in membrane potential (shorten in depolarization, elongate in hyperpolarization). The electromotility of Prestin protein is a force driving the cochlear amplifier to enhancing auditory sensitivity by 100-fold, which amplifies movement of basilar membrane in an active feedback mechanisms. The active feedback mechanism tunes the auditory response to sound stimuli, allowing exquisite sensitivity, extensive range, sharp frequency selectivity in ears of mammals. A report demonstrated that targeted gene knockout directly at prestin caused shifting upward in hearing threshold by 40-60dB SPL in mice. Works indicate that there are two functional domain in the Prestin protein:(1) "Voltage sensor", intracellular monovalent positive anion (chloride) binds to the binding site of prestin as extrinsic voltage sensor of Prestin.(2) "Actuator", a regulator which promote cells to contract or elongate in response to membrane potential in depolarization or hyperpolarization by changing conformation of Prestin protein.After binding to a site with molecular affinity, intracellular chlorides are translocated across the membrane by the transmembrane voltage in a normal cell: toward the extracellular surface upon hyperpolarization, toward the cytoplasmic side in response to depolarization. This translocation triggers conformational changes of the protein that finally change its surface area in the plane of the plasma membrane. In a word, its surface area increases upon hyperpolarization(cell elongation); the area decreases upon depolarization(cell contraction).Prestin acts as an incomplete transporter. It swings anions across the membrane, but does not allow these anions to dissociate and escape to the extracellular space. When monovalent anions are not present in the cytoplasm, all prestin molecules are in their’short’ state, as the OHC is completely contracted. It has been demonstrated that salicylate acts as a competitive antagonist at the anion-binding site of prestin, and the binding affinity of salicylate is around 300-fold higher than that found for Cl- ion, which provides a possible explanation for the significantly reduced OHC electromotility that probably underlies the hearing loss induced by large doses of aspirin.Studies assumed that stereocili located at the top of OHC disorder, loss, and OHCs died in apoptosis or necrosis induced by ototoxicity of AmAn, which cause hearing loss. Therefore we have hypothesis, ototoxicity of AmAn direct affects prestin protein located at basolateral membrane of OHC and causes the expression or functions of prestin protein. This directly effect probably produces direct effects on prestin protein prior to damages in OHC induced by AmAn, which will arise us to renew acknowledges about mechanisms of AmAn ototoxicity.Objectives(1) To establish mic ototoxicitial model induced by kanamycin.(2) To investigate effects of kanamycin induced ototoxicity on OHCs and motor protein Prestin expreesed in basolateral membrane of OHCs, in the mice ototoxicitial model.(3) To study apoptosis of cochlear tissue and cells, apoptosis signaling pathway and its characteristics during the course induced by kanamycin.(4) To study the effects of kanamycin and extracellular chloride on membrane potential and intracellular chloride in CHO-K1 cells stably transfected with Prestin gene to further evaluate their effects on functions of prestin.Methods1、Ototoxicity model in BALB/c mice was induced by kanamycin.2、Measurement of ABR:To detect changes of hearing thresholds in different frequency of tone-pip stimuli sound, evaluate the changes of hearing function, and the difference between kanamycin administration groups and control group.3、Immunohistochemistry:Damages of OHCs and effects on prestin protein induced by ototoxicity were studied with the methods of cochlear paraffin sections and combine of cochlear basilar membrane whole tissue mounting, immunofluorescence staining in situ, laser scanning confocol microscope imaging techniques, correlation between express of motor protein prestin in cochlear outer hair cells and ototoxicity was studied. Paraffin sections, TUNEL assay and immunohistochemistry methods were used to investigate apoptosis, apoptosis signaling pathway, and apoptosis characteristics in cochlear cells during the course of ototoxicity induced by kanamycin.4、Real-Time PCR testing:The expression levels of Prestin RNA in kanamycin administration groups and control group were measured to investigate the effect of kanamycin on prestin protein at molecular level. Expressions of miR-34a, miR-34c in cochlea in mice treated with kanamycin measured and confirm whether both miRNA involve in regulation of cells apoptosis in cochlea induced by AmAn ototoxicity.5、Prestin-pcDNA3.1(-) plasmids were stably transfected into CHO-K1 cells, and the clone with high prestin expression level was selected by selection reagent. Fluorescence dye probes and microscope fluorescence image recording were applied to analysis effects of kanamycin and extracellular chloride on membrane potential and intracellular chloride in cells expressing prestin protein and in control group. To study relation between Prestin protein and cells apoptosis with measurement of cells apoptosis induced by kanamycinResults1、Ototoxicity model in BALB/c induced by kanamycin was successfully established.2、During the ototoxicitial course induced by kanamycin in mice, the thresholds of ABR in each stimuli frequency shifted upward in a time-dependent manner in drug-administrated mice, whereas there is no significant change in hearing thresholds of ABR in the control group. There are significant differences in hearing thresholds of ABR between the drug-treated group and the control groups after 10 days treatment. The thresholds of high frequency ABR shifted (≥24kHz) (after 7 days) earlier than those in lower frequencies. (≤8kHz).3、The degree of damages in cochlear outer hair cells was time-dependent; The expression level and locations of motor protein prestin changed as well. The RNA level of prestin was decreased and showed a negative correlation with the duration of kanamycin administration.4、The results from TUNEL assays of cochlear paraffin sections of drug-treated groups showed that cochlear cells underwent apoptosis that was, to some extent positively correlated with the duration of kanamycin administration.5、Signal transduction pathways underlying cell apoptosis in cochlea induced kanamycin -treatment were investigated with immunohistochemistry and the results showed that calpain apoptosis signaling pathwaywas predominant, and the protein level of calpain increased with the duration of drug treatment.6、Real-time PCR showed that the level of miR-34a involved in regulation of cochlear cells apoptosis was increased with the dosage of kanamycin, while the level of another small RNA involved in cells apoptosis-miR-34c showed no significant difference between the drug-treatment and control groups.7、Intracellular chloride concentration was increased and membrane potential was depolarized upon kanamycin treatment in CHO-K1 cells expressing high levels of prestin protein. Removal all extracellular chlorides could also result in depolarization and slight increase of intracellular chloride concentrations. Comparing between prestin-tranfected and the control groups, membrane potential changes induced either by kanamycin treatment or removal of extracellular chlorides showed no significant difference. Effects of removal of extracellular chlorides on intracellular chloride concentrations were not significantly different between the two groups either. However, kanamycin treatment affected intracellular chloride concentrations with a significant difference between the two groups.8、Kanamycin treatment can induce cells apoptosis in duration-dependent; Apoptosis rate of cells transfected Prestin was higher signifantly than that of control cellsDiscussionResults of our study showed that hearing thresholds shifted upward in mice model of ototoxicity induced by kanamycin treatment. The thresholds of higher frequencies changed earlier than those of lower frequency, because the cochlea damage induced by kanamycin early appeared first at cochlea basal where sensory cells are more sensitive to high frequency sound stimuli, and then the damages would spread from basal to apex where sensory cells are more sensitive to low frequency sound stimuli. Therefor, high frequency hearing was affected at earlier stage by ototoxicity induced by kanamycin treatment, while low frequency hearing was impacted later.The results indicated that OHCs and other tissue or cells in cochlea underwent apoptosis during chronic kanamycin treatment, and the calpain signaling transduction pathway was a predominant apoptosis pathway in this process. Ototoxicity induced by kanamycin caused increases of intracellular calcium concentration, which activated calpain protein excessively, resulting in the collapsion of cytoskeleton and cellular membrane and changes of membrane proteins, various kinases, phosphotases and transcription factors which eventually lead to cell death. MiR-34a was also involved in regulations of cochlear cell apoptosis and its expression level was positively correlated to administration time of the drug, but the expression level of miR-34c was not affected. MiRNA is a kind of small molecular RNA, which affects gene expression, cell cycle regulation, and development in euccaryotes. MicroRNA is a molecule which triggers RNA interferences in cell proliferations, death, apoptosis and fat metabolism. It also involves in transposable inhibitions, genetic recombination and regulations of gene expressions at transcriptional and post-transcriptional level. Some studies indicated that both miR-34a and miR-34c were involved in cells apoptosis regulation, however, our results indicated that miR-34a was involved in regulation of cochlear cells apoptosis during ototoxicitial course induced by kanamycin, and the expression level of expression of miR-34a was dependent on the duration of drug administration. On the other hand miR-34c did not play a role in this process. How does it generate, and how regulate still be unknown until explanations concluded by further studies.Motor protein prestin located in the basolateral membrane of OHCs was affected in the course of cells apoptosis by ototoxicity induced by kanamycin and the expression level of prestin protein was declined. Morphological results showed that locations of prestin protein were changed in that prestin protein "fell off" from OHCs and aggregated to form clusters in scarring areas that were formed by apoptosis and loss of OHCs. Some prestin protein fragments may enter surrounding supporting cells and the final fate of these protein fragments needs to be further explored. Results from molecular biology showed that the decline of RNA level of prestin (after 7d drug-administration) was positively correlated to drug administration time and prior to OHCs shape change (after 14d drug-administration) and the appearance of abnormal morphology of prestin protein (after 14d drug-administration) in the course of ototoxicity induced by kanamycin, indicating kanamycin may directly affect the regulation of prestin protein expression at molecular level, which in turn influenced the function of prestin protein.Intracellular chloride concentration plays a vital role on functions of prestin. Intracellular chloride can bind to the binding site on the prestin protein, thus causing the conformational changes of prestin protein. Therefore, intracellular chloride is also called "voltage sensor" for prestin. Prestin will lose its function if all intracellular chlorides are removed, indicating intracellular chloride is vital to prestin functions. Hence, the changes of intracellular chloride concentration can directly affect functions of prestin. The change of cell membrane potential is also important to functions of prestin. Upon membrane potential depolarization, prestin protein contracts causing cells shorten, while upon hyperpolarization, prestin elongates which causes cells elongation. Thus functions of prestin protein could be indirectly evaluated by studying effects of kanamycin on membrane potential. Effects of changes of extracellular chloride concentration on cell membrane potential and intracellular chloride concentration in prestin-transfected cells were also studied.Our results showed that kanamycin could directly affect membrane potential by causing membrane potential depolarization, but there was no significant difference between transfection and control groups which indicated this effect of kanamycing on membrane potential was non-specific, which could be due to binding of kanamycing to the membrane which resulted in increased intracellular calcium concentration and subsequent membrane depolarization. In vivo, kanamycing, once in the surrounding area of cochlear OHCs, can sill indirectly affect the functions of prestin by causing membrane potential depolarization, and thus influence hearing function of cochlea. Kanamycing increased intracellular chloride concentration significnatly in CHO-K1 cells expressing high level of prestin protein in high level than in controls. This could be due to the release of chloride bound in the the binding site of prestin protein into cytoplasma either induced by competitive inhibition of kanamycin, or induced by membrane potential depolarization which resulted in prestin protein "shorten". Thereby intracellular chloride concentration changed more in tranfection group than in control. It is also found that the onset of kanamycin’s effect on membrane potential and intracellular chloride concentration were very fast (a few minutes to tens of minutes), which might explain the acute effects of ototoxicity of AmAn in clinical treatments. The acute ototoxicitial effects will gradually disappear and hearing function will recover by discontinuing administration when ototoxicity appears in the early period treatment of AmAn.The changes of membrane potential and intracellular chloride concentration are fast and reversible, which lead us to speculate that cell functions will recover once the contributed factors are eliminated. Therefore, we believe, that acute ototoxicitial effect of AmAn probably is due to effects induced by AmAn on inner ear homeostasis, particularly on outer hair cells, which cause prestin protein located in outer hair cells membrane to function abnormally. However, the acute ototoxicity effect is quick and reversible, and dysfunction can recover, OHCs shape and hearing function come back to normal when AmAn in inner ear is gradually dismished. As for ototixicity induced by long term administration of AmAn, the drug could activate some stress mechanisms, such as cell apoptosis pathway, outer hair cell death in apoptosis pathway and loss, causing changes of locations of prestin protein, declines in level of prestin expression, and abnormal functions of prestin, which eventually lead to irreversible damages in hearing function.In a word, AmAn directly affected prestin protein functions prior to damages of OHCs in the ototoxicitial course. AmAn also directly affects RNA synthesis of prestin, thus regulating expression and functions of prestin protein, declines hearing function, causes acute ototoxicity. The effect of acute ototoxicity appears fast and reversible. Hearing function will gradually recover by removal the effects of AmAn in time; if not, OHC died in apoptosis and lost, expression of prestin declined significantly, location of prestin changed induced by ototoxicity of AmAn, hearing function was damaged irreversible. The results are vital referent values for clinical preventing and curing ototoxicity of AmAn. Auditory functions should be monitored in short interval time when long-term administration of AmAn in patients. Hearing functions in high frequency (12 kHz to 16 kHz) which easily influenced by acute ototoxicity should be detected. Administration should be adjusted or discontinued for some time when the function appeared disorder early, and treatment could be continued when the auditory functions recover.Conclusions1、Chronic ototoxicity was induced by long-term kanamycin administration in mice model, and the functional damages of hearing aggravated with extension of treatment, hearing thresholds shifted upward, high frequency hearing function was damaged first. 2、Cochlear cells died in apoptosis pathway, which was dependent on treatment duration following mice ototoxicity induced by kanamycin.3、During cochlear cells apoptosis, calpain signaling transduction pathway was predominant and the up-regulation of expression of calpain protein was positively correlated with treatment duration.4、MiR-34a was involved in the regulation of cochlear cells apoptosis induced by kanamycin, and the expression level of miR-34a was positively correlate to administration time and level of cells apoptosis.5、Chronic ototoxicity induced by kanamycin caused OHCs collapse, death, and loss, resulted in decline of expression of prestin protein in a time-dependent manner and changes of locations of prestin protein. Kanamycin could directly affect RNA synthesis of prestin protein, and RNA contents of prestin protein decreased gradually with the extension of drug administration. Change of RNA content appeared earlier than the morphological changes of OHCs and prestin protein expression.6、Results of our studies showed that kanamycin or changes of extracellular chloride concentration could cause membrane potential depolarization non-specifically in both CHO-K1 cells expressing high level of prestin protein and in control cells. Kanamycin increased intracellular chlorides of cells significantly in cells expressing high level of prestin protein. Membrane potential depolarization and changes of intracellular chloride can both affect the functions of prestin located in membrane.7、Kanamycin can induce cell apoptosis, apoptosis rate in Prestin transfected cells was significantly higher than that in control cells. |
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