Craig Whitworth

Mechanisms of cisplatin-induced ototoxicity and prevention.

Cisplatin is a widely used chemotherapeutic agent to treat malignant disease. Unfortunately, ototoxicity occurs in a large percentage of patients treated with higher dose regimens. In animal studies and in human temporal bone investigations, several areas of the cochlea are damaged, including outer hair cells in the basal turn, spiral ganglion cells and the stria vascularis, resulting in hearing impairment. The mechanisms appear to involve the production of reactive oxygen species (ROS), which can trigger cell death. Approaches to chemoprevention include the administration of antioxidants to protect against ROS at an early stage in the ototoxic pathways and the application of agents that act further downstream in the cell death cascade to prevent apoptosis and hearing loss. This review summarizes recent data that shed new light on the mechanisms of cisplatin ototoxicity and its prevention.

Ototoxicity: therapeutic opportunities

Two major classes of drugs currently in clinical use can cause permanent hearing loss. Aminoglycoside antibiotics have a major role in the treatment of life-threatening infections and platinum-based chemotherapeutic agents are highly effective in the treatment of malignant disease. Both damage the hair cells of the inner ear, resulting in functional deficits. The mechanisms underlying these troublesome side effects are thought to involve the production of reactive oxygen species in the cochlea, which can trigger cell-death pathways. One strategy to protect the inner ear from ototoxicity is the administration of antioxidant drugs to provide upstream protection and block the activation of cell-death sequences. Downstream prevention involves the interruption of the cell-death cascade that has already been activated, to prevent apoptosis. Challenges and opportunities exist for appropriate drug delivery to the inner ear and for avoiding interference with the therapeutic efficacy of both categories of ototoxic drugs.

Dose-Dependent Protection by Lipoic Acid against Cisplatin-Induced Nephrotoxicity in Rats: Antioxidant Defense System

This study was designed to investigate the role of graded doses of lipoic acid pretreatment against cisplatin-induced nephrotoxicity. Male Wistar rats were divided into six groups and treated as follows: 1) vehicle (saline) control; 2) cisplatin (16 mg/kg, intraperitoneally); 3) lipoic acid (100 mg/kg, intraperitoneally); 4) cisplatin plus lipoic acid (25 mg/kg); 5) cisplatin plus lipoic acid (50 mg/kg) and 6) cisplatin plus lipoic acid (100 mg/kg). Rats were sacrificed three days after treatment, and plasma as well as kidneys were isolated and analyzed. Plasma creatinine increased (677% of control) following cisplatin administration alone which was decreased by lipoic acid in a dose-dependent manner. Cisplatin-treated rats showed a depletion of renal glutathione (GSH), increased oxidized GSH and decreased GSH/GSH oxidized ratio (62%, 166% and 62% of control), respectively which were restored with lipoic acid pretreatment. Renal superoxide dismutase, catalase, glutathione peroxidase (GSH peroxidase) and glutathione reductase activities decreased (62%, 75%, 62% and 80% of control), respectively, and malondialdehyde content increased (204% of control) following cisplatin administration, which were restored with increasing doses of lipoic acid. The renal platinum concentration increased following cisplatin administration, which was possibly decreased by chelation with lipoic acid. The data suggest that the graded doses of lipoic acid effectively prevented a decrease in renal antioxidant defense system and prevented an increase in lipid peroxidation, platinum content and plasma creatinine concentrations in a dose-dependent manner.

Application of Antioxidants and Other Agents to Prevent Cisplatin Ototoxicity

Objective/Hypothesis: To review the recent data from experiments performed in this laboratory to test the hypothesis that cisplatin ototoxicity is related to depletion of glutathione and antioxidant enzymes in the cochlea and that the use of antioxidants or protective agents would protect the cochlea against cisplatin damage and prevent hearing loss.

Vitamin E Reduces Cisplatin Ototoxicity

Objectives/Hypothesis Cisplatin ototoxicity is a major dose‐limiting factor in the treatment of several neoplasms. Vitamin E, a slow‐acting free radical scavenger, has been shown to ameliorate nephrotoxicity and endothelial cell damage in animals receiving cisplatin. The purpose of the study was to determine the effectiveness of vitamin E as an otoprotectant.

Protection by ebselen against cisplatin-induced nephrotoxicity: Antioxidant system

This study was designed to investigate the cisplatin-induced alteration in renal antioxidant system and the nephroprotection with ebselen. Male Wistar rats were injected with (1) vehicle control; (2) cisplatin; (3) ebselen; and (4) cisplatin plus ebselen. Rats were sacrificed three days post-treatment and plasma as well as kidney were isolated and analyzed. Plasma creatinine increased 598% following cisplatin administration alone which decreased by 158% with ebselen pretreatment. Cisplatin-treated rats showed a depletion of renal glutathione (GSH) levels (52% of control), while cisplatin plus ebselen injected rats had GSH values close to the controls. Antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities decreased 38, 75 and 62% of control, respectively, and malondialdehyde (MDA) levels increased 174% of control following cisplatin administration, which were restored to control levels after ebselen treatment. The renal platinum level did not significantly change with ebselen pretreatment. This study suggests that the protection offered by ebselen against cisplatin-induced nephrotoxicity is partly related to the sparing of antioxidant system.

Potentiation of ototoxicity by glutathione depletion.

The combination of 10 mg/kg ethacrynic acid (ETA) and 100 mg/kg kanamycin (KA) caused neither morphologic damage to the cochlea nor change in the auditory brain stem response of the chinchilla. However, after pretreatment with a single dose of buthionine sulfoximine (BSO; 800 mg/kg intraperitoneally) to reduce intracellular glutathione (γ-glutamylcysteinylglycine; GSH) levels, the above single administration of ETA and KA resulted in complete deafness and severe morphologic damage. The kidney, which has a rapid GSH turnover and is therefore especially susceptible to GSH depletion by BSO, also demonstrated severe damage after this treatment. A similar rapid turnover of GSH and resulting limited capacity to detoxify reactive metabolites and free radicals may determine cochlear and renal vulnerability to this toxicity. These findings may explain the clinical observations of enhanced ototoxicity in patients administered aminoglycoside antibiotics concomitantly with loop diuretics.

Development of endocochlear potential and compound action potential in the rat

The present study was designed to investigate the developmental changes of the endocochlear potential and compound action potential simultaneously from rat pups of various ages. Animals were anesthetized with ketamine/xylazine, and the endocochlear potential was measured with a glass microelectrode. At the same time, a wire electrode was placed on the round window to record the click-evoked compound action potential. The endocochlear potential was found to be very low during the first few days of postnatal life. A rapid increase in the value of the endocochlear potential was noted between eleven and thirteen days of age, and adult-like values were recorded by seventeen days of age. Compound action potential responses were recorded at thirteen days of age to high intensity clicks, followed by a progressive improvement of thresholds and reduction of latencies. The development of the endocochlear potential and compound action potential was found to be reciprocally related - as the magnitude of the endocochlear potential increased, the compound action potential threshold declined with increasing age. The development of the endocochlear potential was found to closely approximate the development of enzymatic activity of sodium, potassium-ATPase in the stria vascularis reported by Kuijpers (1974).

Short Interfering RNA against Transient Receptor Potential Vanilloid 1 Attenuates Cisplatin-Induced Hearing Loss in the Rat

Cisplatin, a chemotherapeutic agent of choice for the treatment of solid tumors, produces hearing loss in approximately half a million new cancer patients annually in the United States. The hearing loss is due, in part, to increased generation of reactive oxygen species (ROS) in the cochlea, leading to lipid peroxidation and damage or death of outer hair cells in the organ of Corti. The cochlea expresses the transient receptor potential vanilloid 1 (TRPV1), which are normally expressed on small diameter neurons in the peripheral nervous system and mediate thermal sensitivity, but whose role in the cochlea is unclear. In this study, we show that TRPV1 is coregulated along with the NADPH oxidase isoform, NOX3, by cisplatin. Induction of these proteins by cisplatin is dependent on ROS generation, since it is reversed by systemic lipoic acid administration. In organ of Corti hair cell cultures (UB/OC-1 cells), cisplatin activates and induces TRPV1 and NOX3, leading to apoptosis of these cells. Inhibition of TRPV1 by capsazepine or ruthenium red reduced the apoptosis, implicating TRPV1 in this process. Treatment of UB/OC-1 cultures with short interfering RNA (siRNA) against either TRPV1 or NOX3 reduced cisplatin-induced apoptosis, while round window application of TRPV1 siRNA to rats reduced TRPV1 expression, decreased damage to outer hair cells and reduced cisplatin-induced hearing loss. These data provide a link between NOX3 and TRPV1 in cisplatin-induced hearing loss and suggest that targeting these proteins for knockdown by siRNA could serve as a novel approach in treating cisplatin ototoxicity.

Up-regulation of adenosine receptors in the cochlea by cisplatin

In a previous study, we have demonstrated the presence of two adenosine receptor (AR) subtypes, namely A1 and A3AR, in the chinchilla cochlea. One or both of these receptors couple to activation of antioxidant enzymes, with resulting decreases in lipid peroxidation. The chemotherapeutic agent, cisplatin, was shown to produce ototoxicity within a few days of administration presumably by generating reactive oxygen species (ROS) and thereby increasing lipid peroxidation. In this study, we focused on whether lipid peroxidation induces hearing loss by assessing the cochlear antioxidant defense system over a shorter time period (24 h) following cisplatin administration. Cisplatin was administered to anesthetized chinchillas by round window membrane application and hearing loss was determined by compound action potential (CAP) and endocochlear potential (EP) 24 and 72 h post-treatment. Elevations in CAP thresholds in response to click and to 2, 4, 8 and 16 kHz tones and decreases in EP were obtained within 24 h of cisplatin treatment. These changes persisted for at least up to 72 h. Measurements of antioxidant enzymes indicate no change in the activities of superoxide dismutase, catalase or glutathione peroxidase, either 24 or 72 h following cisplatin treatment. The levels of malondialdehyde obtained at these time points were equivalent to those obtained from the controls. Furthermore, no difference in cochlear morphology was detectable by scanning electron microscopy at the basal, middle or apical turns of the cochlea within 24 h. By 72 h, however, losses in both inner and outer hair cells were observed in the basal and middle turns of the cochlea. A major finding of this study is that exposure to cisplatin led to a 5-fold up-regulation of [125I]N6-2-[4-amino-3-phenyl]ethyladenosine binding in the cochlea within 24 h, reflecting increases in expression of AR(s) in this tissue. These data indicate a dissociation between cisplatin acute (within 24 h) ototoxicity and lipid peroxidation. Furthermore, up-regulation of AR(s) may represent a rapid compensatory mechanism by the cochlea to counter the toxic effects of increased ROS generated by cisplatin.