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.

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.

Measurement of aortic blood flow by Doppler echocardiography: temporal, technician, and reader variability in normal subjects and the application of generalizability theory in clinical research.

Although Doppler echocardiographic measurements of aortic flow have been found to correlate with stroke volume, the reliability of this technique is unknown. The purpose of this study was to measure the reliability of Doppler estimates of cardiac output by identifying and estimating the magnitude of different sources of error. We measured the reliability of Doppler estimates of cardiac output by identifying the magnitude of sources of error in 11 subjects with studies performed by two technicians and read by two readers. Analysis with generalizability theory demonstrated that the largest portion of the total variance was from differences among patients, with a smaller contribution due to day-to-day variability. Variability due to technician was low for continuous wave Doppler (2.0%), but high for pulsed wave (23.2%). Thus continuous wave, but not pulsed wave Doppler measurements, can be used to detect serial changes in cardiac output due to an intervention.

Measurement of aortic blood flow by Doppler echocardiography: day to day variability in normal subjects and applicability in clinical research.

To assess the reliability of Doppler ultrasound for detecting serial changes in cardiac output in response to experimental interventions, the day to day variability of the minute distance of aortic flow was determined in seven normal subjects maintained in a tightly controlled environment with regard to diet and activities. Measurements were made at the same time on 5 to 6 sequential days from an apical window with use of both continuous wave and pulsed wave Doppler techniques. Two statistical measures of reliability were calculated, the intraclass coefficient of correlation (R), which varies between 0 (null reliability) and +1 (perfect reliability), and the 95% confidence interval for the error-free value of a single measurement. For sequential measurements of arterial pressure, 24 h urinary volume and sodium excretion and body weight, the intraclass coefficients of correlation ranged from 0.85 to 0.99, indicating low day to day variability consistent with tight environmental control. Continuous and pulsed wave modes were proved equally and highly reliable for measuring minute distance of aortic flow. However, continuous wave Doppler ultrasound provided acceptable signals more frequently than did the pulsed wave technique. For continuous wave Doppler ultrasound, R was 0.87 (p less than 0.00001); the 95% confidence interval was +/- 1.81 m/min (or 11% of the mean of all measurements), which indicates that this method can be used in a single individual to detect a greater than 11% change in minute distance measured once before and after an intervention.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-response study of asthmatic volunteers exposed to nitrogen dioxide during intermittent exercise.

Twenty-one mildly asthmatic volunteers were exposed to 0, 0.3, 1.0, and 3.0 ppm nitrogen dioxide (NO2) in purified background air in an environmental control chamber. Exposures were separated by 1-wk periods and occurred in random order. Each lasted 1 hr and included three 10-min bouts of moderately heavy exercise (mean ventilation rate 41 L/min). Exposure temperature was near 22 degrees C and relative humidity near 50%. Specific airway resistance and maximal forced expiratory performance were measured preexposure, after the initial exercise, and near the end of exposure. Bronchial reactivity was assessed immediately following exposure, by normocapnic hyperventilation with subfreezing air. Symptoms were recorded on questionnaires before, during, and for 1-wk after each exposure. Exercise induced significant bronchoconstriction regardless of NO2 level. No statistically significant untoward response to NO2 was observed at any exposure concentration. This negative finding agrees with our previous results, but contrasts with findings elsewhere of respiratory dysfunction after exposure to 0.3 ppm. The discrepancy is presently unexplained, but it may relate to different severity of asthma in different subject groups.

A dose-response study of healthy, heavily exercising men exposed to ozone at concentrations near the ambient air quality standard.

Twenty-four healthy, well-conditioned young adult male volunteers, free of asthma or clinical respiratory allergies, were exposed to purified air containing ozone (O3) at 0.16, 0.14, 0.12, 0.10, 0.08, and 0.00 part per million (ppm). Exposures were separated by 2-week intervals, occurred in random order, and lasted 2 hours each. Temperature was 32 +/- 1 degree C and relative humidity was 38 +/- 3%, simulating Los Angeles area smog conditions. Subjects exercised 15 minutes of each half hour, attaining ventilation rates averaging 68 L/min (approximately 35 L/min per m2 body surface area). Lung function was measured pre-exposure and after 1 hr and 2 hr of exposure. Airway responsiveness to a cold-air challenge was measured immediately following the 2-hr exposure. Symptoms were recorded before, during, and for one-week periods following exposures. For the group as a whole, no meaningful untoward effects were found except for a mild typical respiratory irritant response after 2 hr exposure to 0.16 ppm O3. Two individual subjects showed possible responses at 0.14 ppm, and one of them also at 0.12 ppm. In comparison to some previous investigations, this study showed generally less response to O3. The comparative lack of response may relate to the favorable clinical status of the subjects, the pattern of exercise during exposure, or some other factor not yet identified.

Controlled exposure of volunteers with chronic obstructive pulmonary disease to nitrogen dioxide.

Twenty-two volunteers with chronic obstructive pulmonary disease were exposed to nitrogen dioxide at 0.0, 0.5, 1.0, and 2.0 ppm in a controlled environment chamber. Exposure lasted 1 hr and included two 15-min exercise periods, during which the mean ventilation rate was roughly 16 L/min. Pulmonary mechanical function was evaluated pre-exposure, after initial exercise, and at the end of exposure. Blood oxygenation was measured by ear oximetry pre-exposure and during the second exposure period. Symptoms were recorded during exposures and for 1-wk periods afterward. No statistically significant changes in symptom reporting could be attributed to nitrogen dioxide exposure at any concentration, compared to the 0.0 ppm control condition. Measures of pulmonary mechanics showed either no significant changes, or small and equivocal changes. Arterial oxygen saturation showed marginal improvement with exercise, regardless of nitrogen dioxide concentration.

Respiratory effects of photochemical oxidant air pollution in exercising adolescents.

Healthy volunteers 12 to 15 yr of age (46 boys, 13 girls) were exposed to purified air and to smoggy Los Angeles ambient air on different occasions. The studies were performed in random order approximately 2 wk apart. They included 1 h of continuous bicycle exercise (mean ventilation, 32 L/min) plus brief warm-up and cool-down periods. Symptoms and forced expiratory performance were recorded preexposure in purified air, immediately postexposure, and after 1 h recovery in purified air. Mean exposure temperature was 32 degrees C, and mean relative humidity was 45%. In ambient exposure, pollutant concentrations averaged 0.144 ppm for ozone and 153 micrograms/m3 for total suspended particulates. Group mean FEV1 decreased during ambient exposure (p less than 0.01) and only partially recovered during the following 1 h. Unlike adults studied previously, this subject group reported no significant increase in respiratory symptoms accompanying changes in FEV1. Adolescents may be less aware of early respiratory irritation by oxidants and thus more at risk from ambient exposures than are adults.

Effects of exposure to 4 ppm nitrogen dioxide in healthy and asthmatic volunteers.

Healthy and asthmatic volunteer subjects (N = 25 and N = 23, respectively) were exposed twice each to purified air (control) and to 4 ppm nitrogen dioxide (NO2) in a controlled-environment chamber. Exposures lasted 75 min, and included 15 min each of light exercise (ventilation rate near 25 L/min) and heavy exercise (near 50 L/min). Compared to control, NO2 exposure produced no statistically significant untoward effects on airway resistance, symptoms, heart rate, skin conductance, or self-reported emotional state in normal or asthmatic subjects. Exercise was associated with significantly (P less than .001) increased airway resistance in both subject groups, although the increase in normals was small. In both groups, systolic blood pressure showed small but significant (P less than .01) decreases with NO2 exposure, compared to control. This effect, if real, may relate to formation of a vasodilating nitrite or nitrate from inhaled NO2. The lack of respiratory response contrasts with previous findings elsewhere; at present, this inconsistency is unexplained.

Respiratory responses of humans exposed to an aerosol-gas pollutant mixture: multivariate contrast of a complex atmosphere to clean air and sodium chloride aerosol controls.

Data from a group of 20 subjects with normal baseline pulmonary function, who were exposed for 2 h to a test atmosphere containing a complex mixture of pollutants, have been contrasted with data from two other groups exposed to presumably non-toxic control atmospheres. Group 1 was exposed to clean air, group 2 was exposed to clean air containing sodium chloride aerosol at 270 micrograms m-3, and group 3 was exposed to the complex atmosphere containing sodium chloride (332 micrograms m-3) and zinc ammonium sulfate (23 micrograms m-3) aerosols plus nitrogen dioxide (0.5 ppm) and sulfur dioxide (0.5 ppm). These atmospheres (ranked according to the presumed relative toxicities of the components; clean air = 0, sodium chloride = 1, complex mixture = 2) were contrasted using multiple regression and partial correlation analyses. The effects of exposure to the complex gas-aerosol mixture on forced expiratory performance were not significantly different from those observed in subjects exposed to clean air or to sodium chloride aerosol.

Asthmatics' responses to 6-hr sulfur dioxide exposures on two successive days.

Asthmatic volunteers (N = 14) aged 18 to 33 yr with documented sensitivity to sulfur dioxide (SO2) were exposed in a chamber to 0.6 ppm SO2 for 6-hr periods on 2 successive days. Similar exposures to purified air, 1 wk later or earlier, served as controls. Subjects exercised heavily (target ventilation rate 50 L/min) for 5 min near the beginning of exposure (early exercise) and for an additional 5 min beginning after 5-hr of exposure (late exercise). At all other times, they rested. Body plethysmographic measurements and symptom questionnaires were administered pre-exposure, after each exercise period, and hourly during rest. Bronchoconstriction and lower respiratory symptoms were observed during or immediately following exercise--to a slight extent with clean air, and to a more marked extent with SO2. Bronchoconstriction and symptoms were modestly less severe on the second day of SO2 exposure than on the first day, but there were no meaningful differences in response between early and late exercise periods on either day.

Comparative effects of sulfur dioxide exposures at 5 degrees C and 22 degrees C in exercising asthmatics.

Either airway cooling or sulfur dioxide (SO2) can induce bronchoconstriction in many asthmatics. Whether these two stresses act synergistically is a question with important public health implications. Eight young adult asthmatic volunteers were exposed to SO2 at 0.0, 0.2, 0.4, and 0.6 ppm, during 5 min heavy exercise at 5 degrees C, both with high (approximately 85%) and with low (approximately 50%) relative humidity. Physiologic response increased with increasing SO2 concentration but did not vary significantly with humidity. Symptom response was marginally greater at low than at high humidity. Twenty-four asthmatics were exposed similarly to clean air and to 0.6 ppm SO2, at 5 degrees C and also at 22 degrees C, always at high relative humidity. For this group, physiologic and clinical responses to SO2 (in excess of responses to clean air) were highly significant, regardless of temperature. The mean excess responses at 5 degrees compared with those at 22 degrees C were not statistically significant in clean air or SO2. Thus, moderate cold stress exacerbated the untoward response to SO2 only slightly and inconsistently in these asthmatic subjects.

Response to ozone in volunteers with chronic obstructive pulmonary disease.

Twenty-eight volunteers with chronic obstructive pulmonary disease were exposed to 0.0, 0.18, and 0.25 ppm ozone in purified air for 1-hr periods with light intermittent exercise, with exposure conditions presented in random order at 1-month intervals. No statistically significant changes attributable to ozone were found in forced expiratory performance or percent oxyhemoglobin (measured near the beginning and end of each exposure). No ozone-related changes in clinical status were found by interviews that included the time for 1 wk before to 1 wk after each exposure, except that a moderate increase in lower respiratory symptoms was reported by nonsmokers in 0.18 ppm exposures only. Thus, a slight decrement in hemoglobin saturation with ozone exposure (reported in two previous studies of chronic obstructive pulmonary disease subjects) may not be a common occurrence under typical ambient exposure conditions.

Respiratory effects of 0.75 ppm sulfur dioxide in exercising asthmatics: influence of upper-respiratory defenses.

To determine the influence of mouthpiece breathing on respiratory responses to sulfur dioxide (SO2), 23 young adult asthmatic volunteers were exposed in a chamber to 0.75 ppm SO2 during heavy exercise, once with breathing unencumbered and once while they wore noseclips and mouthpieces. These conditions (more severe than in typical ambient exposures) were deliberately chosen to produce significant physiological and clinical responses. Similar exposures to clean air served as controls. Exposure studies were separated by 1-week intervals and order was randomized. The protocol consisted of 10 min on a bicycle ergometer (mean load 650 kg-m/min, mean ventilation 40 liter/min), preceded and followed by response testing (body plethysmography, symptom questionnaires, and forced expiratory function tests; the last were performed only postexposure). During clean-air exposures, specific airway resistance (SRaw) and symptoms increased significantly, but no meaningful differences between mouthpiece breathing and unencumbered breathing were observed. Exposures to SO2 under these relatively severe conditions produced greater increases in SRaw than clean-air exposures regardless of the mode of breathing, but the excess increase was significantly greater with mouthpiece than with unencumbered breathing. Symptom changes and postexposure forced expiratory function showed qualitatively the same pattern of decrements with SO2 ad did SRaw, but the excess responses attributable to mouthpiece breathing did not attain statistical significance. Mouthpiece breathing can compromise upper-respiratory defenses against SO2 to the extent that responses are greater than with more natural breathing. The mode of breathing should be taken in account when applying laboratory human exposure data to air-quality risk assessment.

Respiratory effects of sulfur dioxide in heavily exercising asthmatics. A dose-response study.

Twenty-three asthmatic volunteers 19 to 31 yr of age were exposed to 0 (control), 0.2, 0.4, and 0.6 ppm sulfur dioxide (SO2) in random order at 1-wk intervals. Exposures took place in a controlled-environment chamber at 23 degrees C and 85% relative humidity; they included 5 min heavy exercise (mean minute volume, 48 L) plus time for postexercise physiologic testing. Body plethysmography (preexposure and end-exposure), spirometry (end-exposure only), and symptom questionnaires (covering the exposure period and the following week) all showed highly significant trends toward increased response with increasing SO2 concentration. Pairwise statistical comparisons showed substantial, highly significant, changes at 0.6 ppm, relative to control. Fewer and smaller significant changes were found at 0.4 ppm. At 0.2 ppm, no significant physiologic changes were found, but increases in symptoms during exposure were possibly significant. Symptom reports 1 day and 1 wk postexposure showed no significant variation related to SO2 level, i.e., exposure-related symptoms apparently reversed in less than 1 day.

Short-term respiratory effects of 0.12 ppm ozone exposure in volunteers with chronic obstructive pulmonary disease.

Twenty-five volunteers with chronic obstructive pulmonary disease of mild to moderately severe degree underwent 1-h exposures to 0.12 ppm ozone (O2) in purified air with intermittent mild exercise. Their responses were assessed in terms of forced expiratory performance, ear oximetry, and reported symptoms. Control studied consisted of similar exposures to purified air alone. Control studies were separated from O2 exposures by 1 month, and the order was randomized. All studies took place in a controlled-environment chamber, and were preceded by approximately 1 h of rest in a purified-air environment. No significant disturbances in forced expiratory performance or symptoms attributable to O2 exposure were found. A slight but significant tendency to decreased arterial hemoglobin oxygen saturation (SaO2) during exercise in O2 was observed. The decrement in SaO2 with O2 relative to clean air (mean 1.3%) was near the limit of resolution of the ear oximeter test and was detected by signal averaging, thus its physiologic or clinical significance is uncertain.