Cytotoxic effects of dimethyl sulphoxide (DMSO) on cochlear organotypic cultures

Abstract

The amphipathic molecule dimethyl sulphoxide (DMSO) is a solvent often used to dissolve compounds applied to the inner ear; however, little is known about its potential cytotoxic side effects. To address this question, we applied 0.1–6% DMSO for 24 h to cochlear organotypic cultures from postnatal day 3 rats and examined its cytotoxic effects. DMSO concentrations of 0.1% and 0.25% caused little or no damage. However, concentrations between 0.5% and 6% resulted in stereocilia damage, hair cell swelling and a dose-dependent loss of hair cells. Hair cell damage began in the basal turn of the cochlea and spread towards the apex with increasing concentration. Surprisingly, DMSO-induced damage was greater for inner hair cells than outer hair cell whereas nearby supporting cells were largely unaffected. Most hair cell death was associated with nuclear shrinkage and fragmentation, morphological features consistent with apoptosis. DMSO treatment induced TUNEL-positive staining in many hair cells and activated both initiator caspase-9 and caspase-8 and executioner caspase-3; this suggests that apoptosis is initiated by both intrinsic mitochondrial and extrinsic membrane cell death signaling pathways.

Introduction

Dimethyl sulphoxide (DMSO), an amphipathic solvent soluble in both water and organic substances, is often used to dissolve hydrophobic substances used in biological research. DMSO has a number of important properties that make it useful clinically. DMSO has been used successfully to treat urinary (McCammon et al., 1998), pulmonary (Iwasaki et al., 1994), rheumatic (Morassi et al., 1989) and dermatological disorders (Burgess et al., 1998). Because of its anti-inflammatory and anti-oxidant properties, DMSO has also been used with some success to treat gastrointestinal disorders (Salim, 1992a, Salim, 1992b). Since DMSO can act as a free-radical scavenger and can cross the blood–brain barrier, it has also been used to treat brain edema (Broadwell et al., 1982, Ikeda and Long, 1990). Although clinically beneficial in some situations, DMSO can have systemic side effects such as diarrhea, vomiting, bronchospasm, hypertension, and pulmonary edema (Davis et al., 1990, Hameroff et al., 1983, Smith et al., 1987, Stroncek et al., 1991). These effects appeared to be dose-dependent (Davis et al., 1990, Stroncek et al., 1991).

The effects of DMSO on cellular function have been studied in a large number of cell types, but with variable results. Some studies have shown that DMSO blocks the rise of intracellular calcium induced by different agents (Reynaud et al., 1999, Saldanha et al., 2002, Santos et al., 2002, Zhang and Eyzaguirre, 1999). Others have reported that DMSO causes an increase in extracellular sodium, potassium and calcium (Santos et al., 2002). DMSO has diverse effects on ion transporters and pumps (Santos et al., 2002). In mouse lymphoma cells, 18 h treatment with 2.5% DMSO induced an apoptotic response consisting of a decline in Bcl-2, a decrease in the mitochondrial membrane potential, the release of cytochrome c from mitochondria, activation of initiator caspase-9 and executioner caspase-3 (Liu et al., 2001). Studies with lymphoma cells suggest that DMSO concentrations of 1–2% prevent apoptosis whereas higher concentrations induces apoptosis (Lin et al., 1995). DMSO enhances the suppressive effects on lidocaine on synaptic transmission (Somei et al., 1995). DMSO (10%) is often used as a cryoprotectant to optimize cell survival in vitro and in vivo (Davis et al., 1990, Trumble and Whalen, 1992). However, when used in vivo DMSO did not protect peripheral nerves from cryoinjury and exacerbated functional recovery (Trumble and Whalen, 1992).

Several studies have used DMSO to dissolve otoprotective compounds that are given systemically or applied locally to the middle ear or round window membrane of the cochlea. NMDA receptor antagonists, in the form of maleate and tartrate salts (metal chelators) dissolved in DMSO and administered systemically protected against aminoglycoside ototoxicity (Basile et al., 1996). However, when sodium maleate or tartaric acid dissolved in DMSO (40%) were given without the NMDA receptor antagonists, they also provided significant protection against aminoglycoside damage (Sha and Schacht, 1998). These findings suggested that the metal-chelating and anti-oxidant properties of these agents acting alone or in combination were protecting against aminoglycoside ototoxicity. Another study infused Texas Red and DMSO into the middle ear space as a control in order to study the distribution of the tracer within the inner ear (Liu et al., 2006). The abstract did not mention the concentration and volume of DMSO applied to the round window or if DMSO caused any cochlear damage. Src protein tyrosine kinase inhibitors dissolved in DMSO (<0.5%) have been placed on to the round window to determine if they would protect against noise induced hearing loss (Harris et al., 2005). No negative side effects from the solvent were reported.

Others have dissolved compounds in DMSO in order to facilitate the entry of these compounds into isolated hair cells (Canlon and Dulon, 1993, Szonyi et al., 1999, Szonyi et al., 2001). One percent DMSO applied to acutely isolated outer hair cells (OHC) resulted in a significant decrease in OHC electromotility (Canlon and Dulon, 1993); the reduction in electromotility was reversed by increasing the concentration of ionomycin, a calcium ionophore (Szonyi et al., 2001). These results indicate that low concentrations of DMSO can exert an immediate effect on OHC function.

Although DMSO is often used to dissolve compounds that are applied to the inner ear in vivo and in vitro, the physiologic and anatomical effects of DMSO on the cochlea are poorly understood. Since DMSO could conceivably cause negative side effects, we applied different concentration of DMSO to cochlear organotypic cultures to evaluate its cytotoxic effects on cochlear organotypic cultures. In contrast to in vitro studies, the cochlear culture preparation offers precise control of the amount of DMSO that can be applied to the inner ear.