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

peer reviewedNumerous 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.

peer reviewedEpidemiological 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.

peer reviewedDespite 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 high‑potassium (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

Étude de la vestibulotoxicité périphérique des solvants aromatiques par une approche in vitro

Aromatic solvents (AS) are chemicals widely used in many industrial sectors. Some solvents can cause balance disturbances, which could be the cause of falls and occupational accidents. It is now well established that AS can damage cochlear cells, but their effects on the vestibular receptor, which shares structural homology with the cochlea, remain unknown. The objective of this thesis is to evaluate the toxicity of various AS on the vestibular receptor using an in vitro model. For this, utricles and ampullas are harvested from newborn rats and cultured in a gelatinous matrix. In this environment, the explants close themselves, inflate and ultimately form a sphere filled with a high-potassium endolymph-like fluid. These 3D cultures conserve all the cells involved in the K+ cycle, the role of which is critical in vestibular physiology. The K+ concentration is measured using a microelectrode filled with an ion-exchange membrane before performing histological analyses. The functionality of different cell types was validated using pharmacological agents capable of inhibiting potassium transporters involved in the K+ cycle such as ouabain, bumetanide, gentamicin and gadolinium. The utricle and ampulla cultures were exposed to a range of styrene concentrations for either 2 h or 72 h. These two types of exposure led to a decrease in the K+ concentration and histological damage in the sensory and secretory epithelia. Changes in K+ concentration seem to be a more sensitive marker of toxicity than histological damage. Then the vestibulotoxic potentials of ethylbenzene, toluene or each of the three xylene isomers were assessed by exposing the utricle cultures to 0.75 mM for 2 h or 72 h. Among the tested solvents, only ethylbenzene and o-xylene caused a decrease in K+ concentration following prolonged exposure, but only o-xylene caused histological damage. Immunohistological analyzes and ATP assays both indicate that AS trigger a necrotic process, rather than an apoptotic sequence. These results show that the chemical structure would be a fundamental parameter in the toxicity of solvents on this model. In conclusion, we suggest that utricle and ampulla cultures could be used to screen many potentially vestibulotoxic molecules rapidly, while using few animals given the eight epithelia available in each newborn rat. This model also facilitates the study of toxic mechanisms on the vestibular receptor.Les solvants aromatiques (SA) sont des produits chimiques très utilisés dans les secteurs industriels. Certains solvants peuvent engendrer des troubles de l’équilibre qui pourraient être à l’origine de chutes et d’accidents professionnels. Il est bien établi que les SA endommagent les cellules cochléaires mais leurs effets sur le récepteur vestibulaire, qui partage une homologie structurelle avec la cochlée, restent à ce jour inconnus. Cette thèse a donc pour objectif d’évaluer la vestibulotoxicité de différents solvants aromatiques présents dans l’industrie à l’aide d’un modèle in vitro. Pour cela, des utricules et des ampoules sont prélevés chez des rats nouveau-nés puis cultivés dans une matrice gélatineuse. Durant la culture, les explants se referment et forment une sphère remplie d’un liquide dont la composition riche en potassium rappelle celle de l’endolymphe. Ces cultures 3D conservent toutes les cellules impliquées dans le cycle du K+ dont le rôle est déterminant dans la physiologie vestibulaire. La concentration en K+ est mesurée à l’aide d’une microélectrode remplie d’une membrane échangeuse d’ions avant analyses histologiques. Des agents pharmacologiques tels que l’ouabaïne, la bumétanide, la gentamicine et le gadolinium, capables d’inhiber les transporteurs potassiques impliqués dans le cycle du K+, ont permis de valider leur fonctionnalité. Les cultures 3D d’utricule et d’ampoule ont été exposées de manière brève (2 h) ou prolongée (72 h) à différentes concentrations de styrène. Ces deux types d’exposition ont baissé la concentration en K+ et provoqué des dommages histologiques dans les épithéliums sensoriel et sécréteur. Les variations de la concentration en K+ semblent être un marqueur de toxicité plus sensible que les dommages histologiques. Ensuite, les potentiels vestibulotoxiques de l’éthylbenzène, du toluène ou des 3 isomères du xylène ont été comparés en exposant les cultures d’utricules à une concentration de 0,75 mM durant 2 h ou 72 h. Parmi les SA testés, seuls l’éthylbenzène et l’o-xylène ont entraîné une baisse de la concentration en K+ suite à l’exposition prolongée, mais seul l’o-xylène a provoqué des dommages histologiques. Les analyses immunohistologiques et les dosages d’ATP indiquent que les SA déclenchent un processus nécrotique plutôt qu’apoptotique. Par ailleurs, les résultats laissent supposer que la structure chimique serait un paramètre déterminant dans la toxicité des SA sur ce modèle. En conclusion, les résultats de cette thèse suggèrent que les cultures 3D d’utricules et d’ampoules permettraient de réduire le nombre d’animaux pour cribler les molécules potentiellement vestibulotoxiques, compte tenu des huit épithéliums disponibles dans chaque rat nouveau-né. Ce modèle facilite également l’étude des mécanismes toxiques sur le récepteur vestibulaire

Study of the peripheral vestibulotoxicity of aromatic solvents by an in vitro approach

Les solvants aromatiques (SA) sont des produits chimiques très utilisés dans les secteurs industriels. Certains solvants peuvent engendrer des troubles de l’équilibre qui pourraient être à l’origine de chutes et d’accidents professionnels. Il est bien établi que les SA endommagent les cellules cochléaires mais leurs effets sur le récepteur vestibulaire, qui partage une homologie structurelle avec la cochlée, restent à ce jour inconnus. Cette thèse a donc pour objectif d’évaluer la vestibulotoxicité de différents solvants aromatiques présents dans l’industrie à l’aide d’un modèle in vitro. Pour cela, des utricules et des ampoules sont prélevés chez des rats nouveau-nés puis cultivés dans une matrice gélatineuse. Durant la culture, les explants se referment et forment une sphère remplie d’un liquide dont la composition riche en potassium rappelle celle de l’endolymphe. Ces cultures 3D conservent toutes les cellules impliquées dans le cycle du K+ dont le rôle est déterminant dans la physiologie vestibulaire. La concentration en K+ est mesurée à l’aide d’une microélectrode remplie d’une membrane échangeuse d’ions avant analyses histologiques. Des agents pharmacologiques tels que l’ouabaïne, la bumétanide, la gentamicine et le gadolinium, capables d’inhiber les transporteurs potassiques impliqués dans le cycle du K+, ont permis de valider leur fonctionnalité. Les cultures 3D d’utricule et d’ampoule ont été exposées de manière brève (2 h) ou prolongée (72 h) à différentes concentrations de styrène. Ces deux types d’exposition ont baissé la concentration en K+ et provoqué des dommages histologiques dans les épithéliums sensoriel et sécréteur. Les variations de la concentration en K+ semblent être un marqueur de toxicité plus sensible que les dommages histologiques. Ensuite, les potentiels vestibulotoxiques de l’éthylbenzène, du toluène ou des 3 isomères du xylène ont été comparés en exposant les cultures d’utricules à une concentration de 0,75 mM durant 2 h ou 72 h. Parmi les SA testés, seuls l’éthylbenzène et l’o-xylène ont entraîné une baisse de la concentration en K+ suite à l’exposition prolongée, mais seul l’o-xylène a provoqué des dommages histologiques. Les analyses immunohistologiques et les dosages d’ATP indiquent que les SA déclenchent un processus nécrotique plutôt qu’apoptotique. Par ailleurs, les résultats laissent supposer que la structure chimique serait un paramètre déterminant dans la toxicité des SA sur ce modèle. En conclusion, les résultats de cette thèse suggèrent que les cultures 3D d’utricules et d’ampoules permettraient de réduire le nombre d’animaux pour cribler les molécules potentiellement vestibulotoxiques, compte tenu des huit épithéliums disponibles dans chaque rat nouveau-né. Ce modèle facilite également l’étude des mécanismes toxiques sur le récepteur vestibulaire.Aromatic solvents (AS) are chemicals widely used in many industrial sectors. Some solvents can cause balance disturbances, which could be the cause of falls and occupational accidents. It is now well established that AS can damage cochlear cells, but their effects on the vestibular receptor, which shares structural homology with the cochlea, remain unknown. The objective of this thesis is to evaluate the toxicity of various AS on the vestibular receptor using an in vitro model. For this, utricles and ampullas are harvested from newborn rats and cultured in a gelatinous matrix. In this environment, the explants close themselves, inflate and ultimately form a sphere filled with a high-potassium endolymph-like fluid. These 3D cultures conserve all the cells involved in the K+ cycle, the role of which is critical in vestibular physiology. The K+ concentration is measured using a microelectrode filled with an ion-exchange membrane before performing histological analyses. The functionality of different cell types was validated using pharmacological agents capable of inhibiting potassium transporters involved in the K+ cycle such as ouabain, bumetanide, gentamicin and gadolinium. The utricle and ampulla cultures were exposed to a range of styrene concentrations for either 2 h or 72 h. These two types of exposure led to a decrease in the K+ concentration and histological damage in the sensory and secretory epithelia. Changes in K+ concentration seem to be a more sensitive marker of toxicity than histological damage. Then the vestibulotoxic potentials of ethylbenzene, toluene or each of the three xylene isomers were assessed by exposing the utricle cultures to 0.75 mM for 2 h or 72 h. Among the tested solvents, only ethylbenzene and o-xylene caused a decrease in K+ concentration following prolonged exposure, but only o-xylene caused histological damage. Immunohistological analyzes and ATP assays both indicate that AS trigger a necrotic process, rather than an apoptotic sequence. These results show that the chemical structure would be a fundamental parameter in the toxicity of solvents on this model. In conclusion, we suggest that utricle and ampulla cultures could be used to screen many potentially vestibulotoxic molecules rapidly, while using few animals given the eight epithelia available in each newborn rat. This model also facilitates the study of toxic mechanisms on the vestibular receptor

Histological analysis of vestibular explants to assess toxicity of chemicals

peer reviewedMany epidemiologic and experimental studies have shown that occupational exposures to aromatic solvents can induce hearing deficits in both humans and laboratory rodents. Although the end-organ of balance, the vestibular labyrinth, is in the inner ear, and has morphological and functional similarities with the cochlea, no information is available on the adverse effects of solvents on this receptor. However, epidemiological data suggest that solvent exposure can generate balance disorders, which might increase the risk of falls. In this paper, we describe the methods for assessing peripheral vestibulotoxicity using cultured vestibular samples (utricles and ampullae) collected from newborn rats. After a few days in culture, these sensory tissues become spheres filled with endolymph, which we call “cysts”. Endolymphatic potassium concentration measurements and histopathological observations were performed to understand the toxic mechanisms and identify the cellular targets of a chemical exposure, an aromatic solvent in this case. The model was tested using molecules with known effects: ouabain, a blocker of Na+/K+ ATPase pumps, gentamicin, a cytotoxic antibiotic for vestibular hair cells, and finally styrene, an aromatic solvent used in industry with well-known cochleotoxic properties