Three-dimensional cultured ampullae from rats as a screening tool for vestibulotoxicity: Proof of concept using styrene.

Numerous ototoxic drugs, such as some antibiotics and chemotherapeutics, are both cochleotoxic and vestibulotoxic (causing hearing loss and vestibular disorders). However, the impact of some industrial cochleotoxic compounds on the vestibular receptor, if any, remains unknown. As in vivo studies are long and expensive, there is considerable need for predictive and cost-effective in vitro models to test ototoxicity. Here, we present an organotypic model of cultured ampullae harvested from rat neonates. When cultured in a gelatinous matrix, ampulla explants form an enclosed compartment that progressively fills with a high-potassium (K+) endolymph-like fluid. Morphological analyses confirmed the presence of a number of cell types, sensory epithelium, secretory cells, and canalar cells. Treatments with inhibitors of potassium transporters demonstrated that the potassium homeostasis mechanisms were functional. To assess the potential of this model to reveal the toxic effects of chemicals, explants were exposed for either 2 or 72 h to styrene at a range of concentrations (0.5-1 mM). In the 2-h exposure condition, K+ concentration was significantly reduced, but ATP levels remained stable, and no histological damage was visible. After 72 h exposure, variations in K+ concentration were associated with histological damage and decreased ATP levels. This in vitro 3D neonatal rat ampulla model therefore represents a reliable and rapid means to assess the toxic properties of industrial compounds on this vestibular tissue, and can be used to investigate the specific underlying mechanisms.

An in vitro model to assess the peripheral vestibulotoxicity of aromatic solvents.

Epidemiological and experimental studies indicate that a number of aromatic solvents widely used in the industry can affect hearing and balance following chronic exposure. Animal studies demonstrated that long-term exposure to aromatic solvents directly damages the auditory receptor within the inner ear: the cochlea. However, no information is available on their effect on the vestibular receptor, which shares many structural features with the cochlea and is also localized in inner ear. The aim of this study was to use an in vitro approach to assess and compare the vestibular toxicity of different aromatic solvents (toluene, ethylbenzene, styrene and ortho-, meta-, para-xylene), all of which have well known cochleotoxic properties. We used a three-dimensional culture model of rat utricles ("cysts") with preserved functional sensory and secretory epithelia, and containing a potassium-rich (K+) endolymph-like fluid for this study. Variations in K+ concentrations in this model were considered as biomarkers of toxicity of the substances tested. After 72 h exposure, o-xylene, ethylbenzene and styrene decreased the K+ concentration by 78 %, 37 % and 28 %, respectively. O- xylene and styrene both caused histopathological alterations in secretory and sensory epithelial areas after 72 h exposure, whereas no anomalies were observed in ethylbenzene-exposed samples. These in vitro results suggest that some widely used aromatic solvents might have vestibulotoxic properties (o-xylene, styrene and ethylbenzene), whereas others may not (p-xylene, m-xylene, toluene). Our results also indicate that variations in endolymphatic K+ concentration may be a more sensitive marker of vestibular toxicity than histopathological events. Finally, this study suggests that cochleotoxic solvents might not be necessarily vestibulotoxic, and vice versa.

Styrene alters potassium endolymphatic concentration in a model of cultured utricle explants.

Despite well-documented neurotoxic and ototoxic properties, styrene remains commonly used in industry. Its effects on the cochlea have been extensively studied in animals, and epidemiological and animal evidence indicates an impact on balance. However, its influence on the peripheral vestibular receptor has yet to be investigated. Here, we assessed the vestibulotoxicity of styrene using an in vitro model, consisting of three-dimensional cultured newborn rat utricles filled with a high­potassium (K+) endolymph-like fluid, called "cysts". K+ entry in the cyst ("influx") and its exit ("efflux") are controlled by secretory cells and hair cells, respectively. The vestibular epithelium's functionality is thus linked to K+ concentration, measured using a microelectrode. Known inhibitors of K+ efflux and influx validated the model. Cysts were subsequently exposed to styrene (0.25; 0.5; 0.75 and 1 mM) for 2 h or 72 h. The decrease in K+ concentration measured after both exposure durations was dose-dependent, and significant from 0.75 mM styrene. Vacuoles were visible in the cytoplasm of epithelial cells from 0.5 mM after 2 h and from 0.25 mM after 72 h. The results presented here are the first evidence that styrene may deregulate K+ homeostasis in the endolymphatic space, thereby altering the functionality of the vestibular receptor.