Calcium imaging in gentamicin ototoxicity: Increased intracellular calcium relates to oxidative stress and late apoptosis

Abstract

Objectives

To estimate intracellular calcium changes in gentamicin (GM) ototoxicity using calcium imaging. To investigate GM-induced physiologic changes in auditory cells including cell viability, apoptosis, and oxidative stress.

Methods

Varying concentrations of GM were applied to the HEI-OC1 cochlear cell line. Calcium imaging tracked changes in intracellular calcium concentration during GM cytotoxicity. Cell viability and intracellular reactive oxygen species (ROS) levels also were measured.

Results

Little change in calcium levels occurred in HEI-OC1 cells exposed to less than 35 mM GM. However, calcium rose continuously in cells exposed to more than 60 mM GM. With administration of intermediate concentrations of 40 or 50 mM GM, calcium increased variably in different cells, returning to baseline in some cases, or rising continuously in others. Upon increase of GM concentration, intracellular calcium concentration and ROS were increased, and cell viability was decreased due to late apoptosis.

Conclusion

This study shows that GM increased intracellular calcium, ROS, and late apoptosis of HEI-OC1 cells derived from cochlear tissue. Increase of intracellular calcium is related to GM-induced apoptosis and oxidative stress. Calcium imaging can be used to determine change of intracellular calcium concentrations and apoptosis in GM ototoxicity.

Introduction

Currently, intratympanic gentamicin (GM) is a major treatment modality for intractable Meniere's disease and other conditions involving peripherally initiated vertigo [1], [2], [3]. A major complication of intratympanic GM revealed by these studies is the risk of sensori-neural hearing loss (SNHL), which occurs in 20% of patients [2], [4], [5]. Through trial and error, vertigo control has improved, but the risk of SNHL is still the primary problem with intratympanic GM therapy. A major complicating factor in the occurrence of SNHL is the concentration of intratympanic GM. The concentration of injected GM varies from 18 mg/mL to 80 mg/mL, and a preparation at a concentration of 26.7 mg/mL (55.9 mM) is used commonly [2], [4], [5]. However, little is known regarding the maximal safe dose of GM, in particular its actions on auditory hair cells whose damage underlies SNHL.

GM ototoxicity can be induced by both apoptosis and necrosis pathway, and GM induced apoptosis is related to intracellular calcium increase. In endoplasmic reticulum (ER), GM blocks ribosomal protein synthesis, and causes ER stress, the unfolded protein response (UPR), and cell cycle arrest. Finally the cell undergoes apoptosis, which is mediated by the classical route of calpains and caspase [6] activated by the release of Ca²⁺ from the ER [7]. Meanwhile, GM enters into cells via endocytosis mediated by the megalin/cubilin complex. The drug mostly accumulates in the lysosomes and ER. In the lysosomes, GM produces membrane destabilization and lysosomal aggregation. Eventually GM causes proteolysis and cellular necrosis through cathepsins [7].

The production of mitochondrial reactive oxygen species (ROS) is caused by GM actions on the respiratory chain [7]. GM also reduces glutathione and superoxide dismutase, important antioxidants in cells. The accumulated GM is released into the cytosol where it acts on mitochondria and activates the mitochondrial pathway of apoptosis, produces ROS and reduces the ATP reserve [7]. Oxidative stress induced by GM causes an increase in calcium influx through the L-type calcium channel [8]. Increase of calcium causes further increases in superoxide production by the mitochondria, which causes further oxidation of L-type calcium channels and further calcium influx into the cells [8], [9]. Thus, GM causes the production of ROS and calcium overload leading to cellular apoptosis.

Calcium overload can disturb various calcium sensitive targets with key cellular functions [10]. Calcium deregulation also disturbs the homeostasis of auditory cells [11]. It has long been recognized that a disruption of calcium homeostasis is related to cellular damage [12]. In GM ototoxicity, calcium overload is induced by both apoptotic process and oxidative stress, and finally plays a role in cellular death [6], [13].

The purpose of this study is to measure changes in intracellular calcium concentration in GM ototoxicity using calcium imaging. We also investigate cell viability, ROS, and the pattern of apoptosis. Increases in intracellular calcium concentration can indirectly reflect the severity of GM ototoxicity and oxidative stress. Tracking these physiological changes in GM ototoxicity can help to determine the severity of GM ototoxicity, and can be used as a reference about choice of a safe and suitable intratympanic GM dose range for intractable vertigo control.