Date of Award

5-1-2015

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Tischkau, Shelley

Second Advisor

Campbell, Kathleen

Abstract

D-methionine (D-met) has demonstrated otoprotection from noise-, aminoglycoside-, and cisplatin-induced hearing loss in animal studies. As a result, D-met is currently progressing through translational "bench to bedside" research. However, D-met's exact otoprotective mechanisms have not been fully elucidated. This study investigated relationships between dose- and time-dependent D-met otoprotection from noise- and aminoglycoside-induced hearing loss. Further, the study correlated protective D-met dose to endogenous antioxidant enzyme activity and lipid peroxidation. Specific aim 1 tested D-met dose response protection by auditory brainstem response (ABR) threshold shift analysis and outer hair cell (OHC) quantification. D-met doses ranging from 25-200 mg/kg/dose were administered to chinchillas every 12 hours five times each before and after steady state noise exposure totaling 10 D-met doses. Results demonstrated optimal, sub-optimal, and supra-optimal bi-phasic D-met otoprotective dose response. Optimal D-met protection from steady state noise occurred at the 50 mg/kg/dose level. OHC quantification confirmed electrophysiological assessment. Specific aim 2 measured D-met rescue protection from steady state noise exposure by ABR threshold shift analysis and OHC quantification. Five intraperitoneal (ip) D-met injections were administered every 12 hours beginning 3, 5, 7, 9, 12, 18, 24, 36, or 48 hours after steady state noise exposure. Results measured full D-met protection when administration began as late as 24 hours after noise secession. Significant partial protection was also measured for the 36 hour delay. OHC quantification confirmed electrophysiological assessment. Specific aim 3 measured D-met preloading protection from steady state noise exposure by ABR threshold shift analysis and OHC quantification. Five ip D-met injections were administered every 12 hours beginning 2, 2.5, or 3 days prior to steady state noise exposure. Results measured significant D-met protection when administration ended as early as 24 hours prior to noise exposure. OHC quantification confirmed electrophysiological assessment. Specific aim 4 tested dose-dependent D-met influence on antioxidant enzyme activity and oxidative stress in steady state noise-exposed chinchillas. One ip D-met injection, ranging from 25 to 200 mg/kg/dose, was administered every 12 hours beginning 2 days prior to steady state noise exposure for a total of 5 injections. Two hours post-noise exposure, animals were sacrificed and serum, liver, and cochleae were collected for endogenous antioxidant analysis. Glutaredoxin 2 (Grx2) was also analyzed 21 days post-noise exposure. Lower D-met doses (25 and 50 mg/kg/dose) increased superoxide dismutase and catalase activity. Glutathione reductase and glutathione peroxidase activities significantly increased with D-met doses but only at high concentrations (200 mg/kg/dose). At 21 days post-noise, Grx2 activity was significantly decreased in liver but greatly increased in the cochlea with high D-met doses (200 mg/kg/dose). The endogenous enzyme studies suggest optimal protective D-met dose determined in specific aims 1 through 3 may be secondary to increased superoxide dismutase and catalase activity which may result from D-met's free radical scavenging characteristics. Glutathione pathway activity increased only with high D-met doses that resulted in less optimal protection in specific aim 1. Thus, D-met-induced glutathione pathway enhancement may be a compensatory or saturation mechanism rather than the primary protective mechanism. Further, the extended pre-loading and rescue protection may be a result of significantly increased s-glutathionylation activity in the cochlea. Specific aim 5 tested D-met protection from impulse noise exposures. D-met dose response, rescue, and antioxidant enzyme assay protocols, similar to those in specific aims 1, 3, and 4 in steady state animals, were performed on impulse noise-exposed chinchillas. D-met provided dose- and time-dependent optimal protection from impulse noise similar to the steady-state noise studies. Optimal D-met protection was measured at the 100 mg/kg/dose level with complete rescue protection as late as 24 hours post-noise exposure. Endogenous enzyme activity measures demonstrated significant superoxide dismutase, catalase, and glutathione peroxidase activity increases which correlated with optimal D-met protective dose (100 mg/kg/dose) and catalase and superoxide dismutase activity decreases at the higher doses (200 mg/kg/dose). Specific aim 6 tested dose-dependent D-met protection from tobramycin, amikacin, kanamycin, and gentamicin aminoglycoside antibiotics. Guinea pig animal models were normalized to achieve a 30-40 dB ABR threshold shift with the lowest possible aminoglycoside dose. D-met and the aforementioned single aminoglycoside were administered for 21, 28, 23, or 14 days, respectively. ABRs were collected and assessed at baseline, 2, 4, and 6 weeks after drug administration initiation. After the 6-week ABR data collection, cochleae were collected and prepared for OHC quantification. ABR threshold shifts and OHC quantifications demonstrate significant bi-phasic D-met-induced protection from each aminoglycoside type with different D-met doses. OHC quantification confirmed electrophysiological assessment. This study identified optimal protective D-met dose for aminoglycoside- and noise- induced ototoxicity. It also identified optimal protective D-met dose timing for steady state and impulse noise-induced hearing loss. Further, this study has identified dose-dependent D-met-induced endogenous antioxidant changes and Grx2 enhancement, and therefore s-glutathionylation, as a potential mechanism for D-met protection. Thus, dose- and time-dependent D-met protection influences endogenous antioxidant activity, but exact optimal D-met protection will continue to warrant further investigation.

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