Mechanisms Of D-Methionine Protective Against Cisplatin-induced Ototoxicity And Vestibulotoxicity

Objective: Cisplatin is a chemotherapeutic agent which is used in thetreatment of solid tumors like ovarian, testicular, lung, head and neck andbladder cancers. Although the treatment of cancers with this drugis effective, dose limiting side effects of cisplatin include nausea,nephrotoxicity, neurotoxicity, and ototoxicity occur during or after thetreatment. The clinical presentation of cisplatin ototoxicity is high-frequencysensorineural hearing loss, permanently and bilaterally, and involves the lowerfrequencies as toxicity progresses. Besides the hearing loss, cisplatin-inducedvestibulotoxicity is also a serious problem. Vestibulotoxicity can cause vertigoand influences patient’s ability of movement.Today, how cisplatin damage the inner ear is not completely known to us.Besides the organ of Corti, cisplatin also affects the spiral ganglion and striavascularis, but there are rare reports on the tissue of vestibule. Reactiveoxygen species (ROS) are considered to be involved in various types ofcochlear injury. ROS can interact with nitric oxide and form peroxynitriteswhich nitrosylate and inactivate proteins. What’s more, ROS can react withpoly unsaturated fatty acids in the bilipid bilayer of the cell membranes togenerate highly toxic aldehyde4-hydroxynonenal (4-HNE) leading to celldeath. This increase in4-HNE has been associated with increased Ca2+influxinto the cell and apoptosis. Connexin26(Cx26) and connexin30(Cx30) are themost important Connexin in the inner ear, which are essential for normalhearing. Mutations of GJB2gene and GJB6gene, encoding the Cx26andCx30, are the most common causes of non-syndromic hearing loss in human,but whether they participate in cisplatin-induced ototoxicity andvestibulotoxicity has not been studied. In addition, D–methionine is aneffective agent to reduce the damage of cisplatin to inner ear. The aim of the study was to show whether D–methionine can protect cisplatin-inducedototoxicity and vestibulotoxicity on the levels of functions, morphology, ROSand apoptosis, and whether D–methionine have an protective effect on Cx26and Cx30if the connexins participate in cisplatin-induced ototoxicity andvestibulotoxicity.Methods:30male Kunming mice were randomly divided into theregroups:①Control group（n=10）②Cisplatin group（n=10）③D–methionine+Cisplatin group（n=10）. The mice of control group were injected saline oncea day, for3days; the mice of cisplatin group were injected15mg/kg ofcisplatin once a day, for3days; the mice of D–methionine+Cisplatin groupwere injected400mg/kg of D–methionine before injected15mg/kg of cisplatin,once a day, for3days. Before the experiment, all animals accepted ABR andVEMP to assess the hearing function and vestibular function, as well as afterthe experiment, and then get the tissue depending on the different experimentmethods. We used HE staining and anti-myosin-VI staining to see the living ofhair cells in the inner ear; immunohistochemistry of4-HNE and TUNELstaining were used to assess the level of ROS and apoptosis; the expression ofCx26and Cx30were examed by immnofluorescence staining and western blot.Results were analyzed statistically with the SPSS13.0to determine thedifferences in this experiment, the data were presented as mean±standarddeviation and statistical significance was determined at P<0.05.Results:1General observations: The mice in the control group grew well, keptmoving and were sensitive to sound; the mice in the cisplatin group were thin,didn’t like moving and weren’t sensitive to sound; the mice in theD–methionine+Cisplatin group grew well, also kept moving and sensitive tosound.2The results of ABR test: There were no differences among the threegroups in ABR test before experiment. After the experiment, the ABRthreshold was （3.25±3.35）dB SPL in control group，（63.10±19.87）dB SPLin cisplatin group，and（41.67±13.17）dB SPL in the D-methionine+cisplatin group, there were differences in any two groups(P<0.05).3The results of VEMP test: There were no differences among the threegroups in VEMP test before experiment. After the experiment, the mice ofcontrol group represented normal waves, and the latencies of Ⅰ, II were(0.57±0.12) ms and (0.89±0.13) ms; the mice of cisplatin group didn’trepresent any waves; the mice of D-methionine+cisplatin group couldrepresent general normal waves, but the latencies of Ⅰ, II were (1.60±0.54)msand (1.98±0.53)ms, much longer than the control group.4The results of HE staining and anti-myosin-VI staining: The hair cellsin the control group arranged orderly, and their structures were seencompletely; a lot of hair cells were lost in the cisplatin group, especially in theouter hair cells; much less hair cell lost were seen in the D-methionine+cisplatin group, and the remainder’s structure was normal.5The results of4-HNE immunohistochemistry: The mice of controlgroup didn’t represent positive results in the basilar membrane, stria vascularis,spiral ganglion and saccule; the positive results in the basilar membrane, striavascularis, spiral ganglion and saccule of cisplatin group were obvious; therewere much less positive results in the D-methionine+cisplatin group thancisplatin group in cocheal and saccule.6The results of TUNEL staining: No TUNEL positive cells were seen inthe basilar membrane, stria vascularis and spiral ganglion, as well as saccule,in the control group; many TUNEL positive cells appeared in the cochealtissue of cisplatin group, especially in the lateral wall, and some positive cellcould also be seen in saccule; TUNEL positive cells were much less inD-methionine+cisplatin group than in cisplatin group in the cocheal tissue,and we didn’t find any positive cells in the saccule of this group.7The results of Cx26and Cx30immnofluorescence staining: Besidessaccule, a lot of Cx26and Cx30protein could be seen in the basilar membrane,stria vascularis and spiral ganglion of control group; and they really decreasedin the cisplatin group, including cocheal and saccule; in the D-methionine+cisplatin group, the immnofluorescence staining of Cx26and Cx30was higher than cisplatin group, but lower than the control group.8The results of Cx26and Cx30western blot test: The expression ofCx26and Cx30in the cisplatin group was much lower than the control groupand D-methionine+cisplatin group, and there were no differences betweenthe control group and D-methionine+cisplatin group.Conclusions:1D-methionine can protect against Cisplatin-induced Ototoxicity andVestibulotoxicity, which can be seen in both functional study andmorphological study.2Reactive oxygen species (ROS) and apoptosis are considered to beinvolved in the inner injury caused by cisplatin, and D-methionine can weakenthe roles of ROS and apoptosis to protect this injury.3Cx26and Cx30may participate in cisplatin-induced ototoxicity andvestibulotoxicity, and D-methionine can prevent their decline induced bycisplatin.