TY - JOUR AU - Pottenger, Lynn, H. AB - Abstract Propylene oxide is a nasal toxicant and weak site-of-contact carcinogen in the mouse and rat. To aid in inhalation risk assessment of this vapor and to provide data for comparison to the rat, the current study was aimed at providing quantitative information on upper respiratory tract (URT) dosimetry of this vapor in the mouse. Toward this end, uptake efficiencies of propylene oxide were measured in the surgically isolated URT of the male B6C3F1 mouse under constant velocity inspiratory flow conditions at flow rates of 12 and 50 ml/min and exposure concentrations of 25, 50, 100, 300, or 500 ppm. URT uptake efficiencies were measured continuously during 1 h exposure; mice were terminated immediately after exposure and nasal respiratory and olfactory mucosal nonprotein sulfhydryl (NPSH) levels were determined. Propylene oxide was scrubbed with moderate efficiency in the URT, with uptake efficiencies of ≤ 33 and ≤ 16% at the low and high inspiratory flow rates, respectively. Uptake efficiencies were slightly (∼ 5%) higher at low (25 or 50 ppm) than high (300 or 500 ppm) exposure concentrations, suggesting that a saturable uptake pathway may exist. Nasal tissue NPSH levels were significantly depleted at exposure concentrations of 300 and 500 ppm but not at concentrations of 100 ppm or lower. Similar levels of NPSH depletion were observed in both nasal respiratory and olfactory mucosa. These data from mouse show some key differences when compared with those reported for the rat. propylene oxide, upper respiratory tract, B6C3F1 mouse, nasal nonprotein sulfhydryls Propylene oxide (1,2-epoxypropane, CAS# 75-56-9) is a chemical intermediate used in the manufacture of polyurethane foams, resins, and propylene glycol. It is highly volatile and moderately soluble with a water:air partition coefficient of 78 (Schmidbauer et al., 1996) and a blood:air partition coefficient of 60 (Lee et al., 2005). Propylene oxide is a site-of-contact carcinogen in rodents, inducing tumors at the application site, including forestomach tumors after intragastric administration in the Sprague-Dawley rat (Dunkelberg, 1982), and local tumors after subcutaneous injection (Dunkelberg, 1981). It is a nasal toxicant (Kuper et al., 1988; Lynch et al., 1984; Renne et al., 1986; Rios-Blanco et al., 2003) and weak nasal carcinogen in the rodent. Chronic exposure to concentrations of 300 ppm or more results in a low incidence (< 7%) of papillary adenomas in the rat nose and hemangiomas (8% incidence), hemangiosarcomas (7%), and adenocarcinomas (2%) in the mouse nose (Lynch et al., 1984; Renne et al., 1986). It is a reactive electrophile and reacts directly with macromolecule nucleophilic sites as well as with nonprotein sulfhydryls (NPSH, Lee et al., 2005). It is detoxified via epoxide hydrolase and glutathione-S-transferase (Faller et al., 2001). Propylene oxide metabolism kinetics for these pathways has been well characterized for the rat (Faller et al., 2001). Blood propylene oxide levels and respiratory tract (nasal and pulmonary) NPSH depletion following propylene oxide exposure have also been well characterized in this species (Lee et al., 2005). Previous studies in this laboratory have examined the upper respiratory tract (URT, defined as all regions of the respiratory tract anterior to and including the larynx) dosimetry of inspired propylene oxide in the F344 rat. The vapor was scrubbed with moderate efficiency (30% or less) in the surgically isolated URT at flow rates approximating one-half to twice the predicted minute ventilation of that species. The moderate scrubbing efficiency was consistent with its moderate blood:air partition coefficient, with steady-state uptake efficiencies being primarily maintained by clearance of propylene oxide vapor from nasal tissues via the circulation (Morris et al., 2004). Similar uptake efficiencies were observed at exposure concentrations ranging from 25 to 500 ppm. One hour exposure of the isolated URT to propylene oxide at exposure concentrations of 50 ppm or more resulted in significant depletion of nasal NPSH levels (Morris et al., 2004) with nasal respiratory mucosal NPSH levels being depleted to levels less than 20% of control at exposure concentrations of 500 ppm. In general, the degree of NPSH depletion observed in the isolated URT studies correlated with the degree observed in whole-body inhalation studies (Lee et al., 2005; Morris et al., 2004). While considerable data are available on dosimetry and disposition of propylene oxide in the URT of the rat, little information is available for the mouse. The current study was aimed at providing such data for the mouse. Specifically, the aims of this study were (1) to provide insights into nasal uptake and disposition of propylene oxide in the mouse, (2) to compare and contrast propylene oxide dosimetry in the rat and mouse, and (3) to provide data for generation of dosimetry models for nasal disposition of propylene oxide. Toward these ends two series of experiments were performed. The first experiments characterized the time course for nasal NPSH depletion. To mimic our previous rat studies (Morris et al., 2004), mice were exposed to 300 ppm propylene oxide for 15, 30, 45, or 60 min and terminated immediately after exposure for nasal respiratory and olfactory mucosal NPSH determination. Concentration-response relationships for propylene oxide uptake and NPSH depletion were examined in the second experiment. Since uptake depends strongly on the inspired flow rate, two inspiratory flow rates were used: 12 and 50 ml/min. These correspond to approximately one-half and twice the predicted minute ventilation rate of the mouse (Morris, 1997). Equivalent inspiratory flow rates were used in our studies on propylene oxide in the rat. Mice were exposed to 25, 50, 100, 300, or 500 ppm propylene oxide to match the concentrations used in the rat studies (Morris et al., 2004). METHODS Animals, surgical procedure, and tissue collection. Male B6C3F1 mice (Charles River, Wilmington, MA, B6C3F1/Crl, 5–6 weeks of age at the time of purchase) were used in all experiments. Animals were housed over hardwood shavings (Sani-Chip Dry, P.J. Murphy Forest Products, Montville, NJ) in animal rooms maintained at 22–25°C with a 12-h light-dark cycle (lights on at 6:30 A.M.). Food (Lab Diet, PMI Nutrition International, Brentwood, MO) and tap water were provided ad libitum. Animals were acclimated 2 weeks prior to use and were used within 6 weeks of arrival. Body weights averaged ∼ 30 g at the time of use. All protocols were approved by the University of Connecticut Institutional Animal Care and Use Committee. For exposure, the URT was surgically isolated by the procedure previously described in detail (Morris, 1999). Briefly, after the onset of urethane-induced anesthesia (1.3 g/kg, ip), a tracheostomy was performed and an endotracheal tube (PE 20 tubing, Clay Adams, Parsippany, NJ) was inserted in an anterior direction until its tip was at the larynx and then was tied in place. Exposures of the surgically prepared mice were performed as described below. Immediately after exposure, each animal was killed by exsanguination via incision of the abdominal vena cava. The vasculature was then perfused with 5 ml saline to minimize blood contamination of nasal homogenates. The skull was removed and split sagitally, and tissues from the nasal cavity were removed to prepare two homogenates (respiratory and olfactory mucosa) as performed in our study on propylene oxide uptake in the rat URT (Morris et al., 2004). The respiratory mucosa homogenate contained tissue from the nasomaxillary turbinates and adjacent septal mucosa; the olfactory mucosa homogenate contained the yellow-tinted olfactory mucosa lining the dorsomedial meatus and the tissues from the ethmoturbinates and adjacent septal mucosa. The respiratory and olfactory mucosa homogenates contained ∼ 0.7 and ∼ 1.9 mg protein, respectively (method of Lowry et al., 1951). The entire collection procedure was completed within 5–10 min after terminal sacrifice. Tissue samples were prepared (Ten Broeck homogenizer) in ice-cold 5% trichloroacetic acid with 3mM EDTA (0.7 ml for respiratory and 1.0 ml for olfactory tissue, respectively) and spun at 10,000 × g for 5 min. The resulting supernatant was stored on ice for subsequent NPSH analysis. Exposure conditions. Mice were exposed in a 0.5-l PVC nose-only exposure chamber. Chamber airflow rates were maintained at 5–10 l/min (depending on the exposure concentration) with clean, heated, and humidified air. Chamber air temperature was maintained between 37–40°C, and water content was approximately 33 mg/l, corresponding to greater than 75% relative humidity at 37°C. The chamber walls and air supply lines were heated to prevent condensation. For generation of atmospheres, propylene oxide was fed via a syringe pump into a glass T tube maintained at approximately 60°C through which 0.8 l/min of air was passed. The vapor-rich air was then passed through a mixing chamber and into the nose-only exposure chamber. Propylene oxide (reagent grade, ≥ 98%) was obtained from Fisher Scientific (Pittsburgh, PA). The exposure chamber and vapor generation apparatus were housed in a fume hood. Uptake measurement. The precise methodology for exposure and measurement of URT uptake efficiency has been described in detail (Morris, 1999). Briefly, the surgically prepared animal was placed in the nose-only chamber in a supine position and the endotracheal tube was connected to an air-sampling line. This sampling system served to draw the chamber air through the isolated URT under constant velocity flow conditions at flows of 12 or 50 ml/min. These flows are within the physiological range for the mouse and correspond to approximately one-half and twice the minute ventilation of the mouse as predicted by the equation of Guyton (see Morris, 1997). During exposure,the animal respired room air (20–24°C, ∼ 50% relative humidity) through the incised trachea. Air was drawn through the sample line directly into a gas-sampling valve of a gas chromatograph (see below). Airflow rates were controlled by rotameters that were calibrated in the sample line with a bubble meter. The sample line was heated to prevent condensation of water vapor. Measurement of uptake efficiency requires determination of the propylene oxide concentration in air entering the URT (Cin) as well as the concentration in air exiting the URT (Cex). Uptake efficiency is calculated from the ratio of the concentrations in these samples (Morris, 1999). To determine Cin, the sampling line was connected directly to the chamber both immediately before and immediately after the animal exposure. The ratio of the before and after Cin concentrations averaged 99.0 ± 4.3% (mean ± SD), indicating that the chamber concentration remained constant. For measurement of Cex, the sample line was connected to the endotracheal tube. Air samples were injected into a gas chromatograph at 3-min intervals (see below) to provide repeated measurement of Cex concentration throughout the exposure. Analytical procedures. Airborne propylene oxide levels were determined by gas chromatography (Varian model 3600 gas chromatograph, flame ionization detection) equipped with a gas-sampling valve as in our previous study on propylene oxide dosimetry (Morris et al., 2004). A 15-m DB-WAX megabore column (Agilent Technologies, Palo Alto, CA) was used with a column temperature maintained at 33°C. The carrier gas (nitrogen) flow rate was 30 ml/min. Under these conditions an entire chromatogram could be completed in 2.5 min. Propylene oxide retention time was 0.2 min. Peak areas were converted to airborne concentration on the basis of standard curves generated by injecting pure propylene oxide (using a cold gas-tight syringe) into 4.3-l glass bottles, allowing 5–10 min for equilibration, and drawing air from the bottle at 12 ml/min through the gas sample valve using chromatographic conditions identical to those used for the animal studies. Standards were prepared daily covering the range of concentrations used in the animal studies; a single composite standard curve comprising all the standards prepared during the study (for which r2 > 0.97) was used to calculate all airborne concentrations. Tissue homogenate supernatants were analyzed spectrophotometrically for NPSH content by the method of Sedlack and Lindsay (1968) using glutathione (Sigma Aldrich, St Louis, MO) as a standard. Control and propylene oxide exposures were performed at the same times of the day to minimize diurnal variation in NPSH content. Data are presented as total NPSH (nmol) content per tissue; the limit of detection corresponded to 3 nmol NPSH per tissue. Statistics. Data are reported as mean ± SEM unless otherwise indicated. Each group contained 8–12 animals. Data were compared by ANOVA followed by the Tukey honest significant difference test. A p < 0.05 was required for significance. Statistical calculations were performed with Statistica software (Statsoft, Tulsa, OK). RESULTS Time Course Study Shown in Figure 1A are nasal respiratory mucosal NPSH levels in mice exposed to 300 ppm for 15, 30, 45, or 60 min, at a flow rate of 50 ml/min. The measured exposure concentration averaged 295 ± 10 ppm (mean ± SD). Respiratory mucosal NPSH levels were similar in control mice exposed to air for 15, 30, 45, or 60 min (p > 0.05, ANOVA); therefore, the data from all control animals were combined to form a single control group. The NPSH level in the combined control group averaged 20 ± 7 nmol (mean ± SD, corresponding to 29 nmol/mg protein). Thus, there was large intra-animal variability as indicated by a standard deviation of approximately 30% of the mean value. NPSH levels differed significantly among exposure groups (p < 0.001, ANOVA, Fig. 1A). NPSH levels were decreased to approximately 50% of control levels by 15-min exposure (p < 0.05, compared to control, Tukey test) and remained at similar levels after 30, 45, or 60 min exposure. FIG. 1. Open in new tabDownload slide (A) Respiratory mucosal NPSH content in rats exposed to 300 ppm propylene oxide for 15, 30, 45, or 60 min at a flow rate of 50 ml/min. Data are expressed as percentage of control and shown as mean ± SEM. Each propylene oxide exposure group contained 8 animals. NPSH levels differed among groups at the p < 0.001 level (ANOVA). Groups with differing letters (a, b) differ significantly (p < 0.05, Tukey test). Control respiratory tissue total NPSH content averaged 20 nmol. Control (air exposed) data are shown at time zero and are pooled data from animals exposed to air for 15, 30, 45, or 60 min. See text for details. (B) Olfactory mucosal NPSH content in rats exposed to 300 ppm propylene oxide for 15, 30, 45, or 60 min at a flow rate of 50 ml/min. Data are expressed as percentage of control and shown as mean ± SEM. Each propylene oxide exposure group contained 8 animals. NPSH levels differed among groups at the p < 0.001 level (ANOVA). Groups with differing letters (a, b) differ significantly (p < 0.05, Tukey test). Control respiratory tissue total NPSH content averaged 59 nmol. Control (air exposed) data are shown at time zero and are pooled data from animals exposed to air for 15, 30, 45, or 60 min. See text for details. FIG. 1. Open in new tabDownload slide (A) Respiratory mucosal NPSH content in rats exposed to 300 ppm propylene oxide for 15, 30, 45, or 60 min at a flow rate of 50 ml/min. Data are expressed as percentage of control and shown as mean ± SEM. Each propylene oxide exposure group contained 8 animals. NPSH levels differed among groups at the p < 0.001 level (ANOVA). Groups with differing letters (a, b) differ significantly (p < 0.05, Tukey test). Control respiratory tissue total NPSH content averaged 20 nmol. Control (air exposed) data are shown at time zero and are pooled data from animals exposed to air for 15, 30, 45, or 60 min. See text for details. (B) Olfactory mucosal NPSH content in rats exposed to 300 ppm propylene oxide for 15, 30, 45, or 60 min at a flow rate of 50 ml/min. Data are expressed as percentage of control and shown as mean ± SEM. Each propylene oxide exposure group contained 8 animals. NPSH levels differed among groups at the p < 0.001 level (ANOVA). Groups with differing letters (a, b) differ significantly (p < 0.05, Tukey test). Control respiratory tissue total NPSH content averaged 59 nmol. Control (air exposed) data are shown at time zero and are pooled data from animals exposed to air for 15, 30, 45, or 60 min. See text for details. Shown in Figure 1B are nasal olfactory mucosal NPSH levels in mice exposed to 300 ppm propylene oxide for 15, 30, 45, or 60 min, at a flow rate of 50 ml/min. Olfactory mucosal NPSH levels were similar in control mice exposed to air for 15, 30, 45, or 60 min (p > 0.05, ANOVA); therefore, the data from all control animals were combined to form a single control group. The total NPSH content in the combined control group averaged 59 ± 16 nmol (mean ± SD, corresponding to 33 nmol/mg protein). As for respiratory mucosa, there was large intra-animal variability as indicated by a standard deviation of approximately 30% of the mean value. NPSH levels differed significantly among groups, were decreased to approximately 50% of control levels after 15 min exposure (p < 0.05, Tukey test), and were at similar levels after 30, 45, or 60 min exposure. URT uptake efficiency was also measured in all mice. Shown in Figure 2 is the propylene oxide URT uptake efficiency in the 60-min exposure group (flow rate of 50 ml/min). Uptake remained steady during the last 30 min of exposure (p > 0.05, repeated measures ANOVA); uptake efficiency averaged 12.6 ± 1.1% during this time period. FIG. 2. Open in new tabDownload slide Propylene oxide uptake efficiency during isolated URT exposure for the 300-ppm exposure group at flow rates of 50 ml/min. Data are expressed as percentage of control and shown as mean ± SEM. Each group contained 8 animals. Uptake efficiency was steady during the last 30 min of exposure (p > 0.05, repeated measures ANOVA) and averaged 12.6%. FIG. 2. Open in new tabDownload slide Propylene oxide uptake efficiency during isolated URT exposure for the 300-ppm exposure group at flow rates of 50 ml/min. Data are expressed as percentage of control and shown as mean ± SEM. Each group contained 8 animals. Uptake efficiency was steady during the last 30 min of exposure (p > 0.05, repeated measures ANOVA) and averaged 12.6%. Concentration-Response Study Shown in Figure 3A are nasal respiratory mucosal NPSH levels in mice exposed to 0 (control), 25, 50, 100, 300, or 500 ppm propylene oxide for 60 min. Measured exposure concentrations averaged 23 ± 2.2, 49 ± 4.1, 108 ± 6.8, 317 ± 15, and 529 ± 25 ppm (mean ± SD) in these exposure groups, respectively. The NPSH data from the 12- and 50-ml/min flow groups at each concentration were combined because two-way ANOVA revealed no significant effect of flow rate on NPSH levels. In control mice respiratory mucosal NPSH content averaged 19.2 ± 11 nmol (mean ± SD, corresponding to 27 nmol/mg protein). NPSH levels differed among groups at the p < 0.0001 level (ANOVA). Respiratory mucosal NPSH was not significantly depleted in the 25-, 50-, or 100-ppm exposure groups (p > 0.05, Tukey test) but was significantly depleted (p < 0.05, Tukey test) in the 300- and 500-ppm exposure groups averaging 51 and 31% of control, respectively, in these groups. The degree of NPSH depletion in the 300-ppm group was similar to that observed in the time course study (see Fig. 1A). FIG. 3. Open in new tabDownload slide (A) Respiratory mucosal NPSH content in rats exposed for 60 min to 0, 25, 50, 100, 300, or 500 ppm propylene oxide. Data are expressed as percentage of control and are shown as mean ± SEM. Data from both flow rates (12 and 50 ml/min) at each exposure concentration were combined; each combined group contained 18–24 animals. Control respiratory mucosa NPSH content averaged 18 nmol. NPSH levels differed among groups at the p < 0.0001 level (ANOVA). Groups with differing letters (a, b) differ significantly (Tukey test, p < 0.05). See text for details. (B) Olfactory mucosal NPSH content in rats exposed for 60 min to 0, 25, 50, 100, 300, or 500 ppm propylene oxide. Data are expressed as percentage of control and are shown as mean ± SEM. Data from both flow rates (12 and 50 ml/min) at each exposure concentration were combined; each combined group contained 18–24 animals. Control olfactory mucosa total NPSH content averaged 64 nmol. NPSH levels differed among groups at the p < 0.0001 level (ANOVA). Groups with differing letters (a, b, c) differ significantly (Tukey test, p < 0.05). See text for details. FIG. 3. Open in new tabDownload slide (A) Respiratory mucosal NPSH content in rats exposed for 60 min to 0, 25, 50, 100, 300, or 500 ppm propylene oxide. Data are expressed as percentage of control and are shown as mean ± SEM. Data from both flow rates (12 and 50 ml/min) at each exposure concentration were combined; each combined group contained 18–24 animals. Control respiratory mucosa NPSH content averaged 18 nmol. NPSH levels differed among groups at the p < 0.0001 level (ANOVA). Groups with differing letters (a, b) differ significantly (Tukey test, p < 0.05). See text for details. (B) Olfactory mucosal NPSH content in rats exposed for 60 min to 0, 25, 50, 100, 300, or 500 ppm propylene oxide. Data are expressed as percentage of control and are shown as mean ± SEM. Data from both flow rates (12 and 50 ml/min) at each exposure concentration were combined; each combined group contained 18–24 animals. Control olfactory mucosa total NPSH content averaged 64 nmol. NPSH levels differed among groups at the p < 0.0001 level (ANOVA). Groups with differing letters (a, b, c) differ significantly (Tukey test, p < 0.05). See text for details. Shown in Figure 3B are nasal olfactory mucosal NPSH levels in mice exposed to 0 (control), 25, 50, 100, 300, or 500 ppm propylene oxide for 60 min. As for the respiratory mucosa, two-way ANOVA did not detect an effect of inspiratory flow rate on olfactory mucosal NPSH levels; therefore, the data from the 12- and 50-ml/min groups for each exposure concentration were combined. In control mice, olfactory mucosal NPSH content averaged 64 ± 16 nmol (mean ± SD, corresponding to 36 nmol/mg protein). NPSH levels differed among groups at the p < 0.0001 level (ANOVA). Although somewhat lower than control levels, olfactory NPSH content did not differ significantly from control levels in the 25-, 50-, or 100-ppm groups. Olfactory NPSH content averaged 63 and 48% of control in the 300- and 500-ppm groups. These values differed from control levels and from each other at the p < 0.05 level (Tukey test). The degree of NPSH depletion in the 300-ppm group was similar to that observed in the time course study (see Fig. 1B). URT propylene oxide uptake efficiencies remained steady during the last 30 min of exposure (Fig. 2p > 0.05, repeated measures ANOVA). The uptake efficiency values obtained during this period were averaged to obtain a single value for each animal for subsequent statistical analysis. Average uptake efficiencies are shown in Table 1. Data at each flow rate were analyzed by ANOVA followed by Tukey test. At a flow rate of 12 ml/min uptake efficiencies differed among exposure concentration groups at the p < 0.01 level (ANOVA); Tukey test revealed that uptake in the 300-ppm group was significantly lower than at 50 ppm. At a flow rate of 50 ml/min, a significant difference among uptake efficiencies in the concentration groups was also observed (p < 0.01, ANOVA), with uptake efficiencies in the 300- and 500-ppm groups being significantly lower than the control group (Tukey test). The deposition rate (nmol/min) was calculated for each animal (as the product of the inspired concentration, the inspiratory flow rate, and the fractional deposition). In the 12-ml/min groups, these rates averaged 4, 8, 14, 33, and 70 nmol/min in the 25-, 50-, 100-, 300-, and 500-ppm groups, respectively. The values, respectively, in the 50-ml/min groups were 8, 15, 28, 66, and 120 nmol/min, about a 2-fold increase in deposition rate for a ∼ 4-fold increase in inspiratory flow rate. TABLE 1 URT Uptake of Propylene Oxide . Exposure groupsa . . . . . . 25 ppm . 50 ppm . 100 ppm . 300 ppm . 500 ppm . 12 ml/min 30b,c ± 1.1 33b ± 1.2 29b,c ± 1.4 26c ± 1.0 29b,c ± 0.9 50 ml/min 16b ± 0.7 15b,c ± 1.0 14b,c ± 0.9 11c ± 0.7 12c ± 1.0 . Exposure groupsa . . . . . . 25 ppm . 50 ppm . 100 ppm . 300 ppm . 500 ppm . 12 ml/min 30b,c ± 1.1 33b ± 1.2 29b,c ± 1.4 26c ± 1.0 29b,c ± 0.9 50 ml/min 16b ± 0.7 15b,c ± 1.0 14b,c ± 0.9 11c ± 0.7 12c ± 1.0 a Uptake was measured continuously throughout the 60-min exposure under constant velocity inspiratory flow conditions at the flow rate indicated for the 25-, 50-, 100-, 300-, or 500-ppm exposure groups. Values represent average uptake efficiency during the last 30 min of exposure and are expressed as percentage of the inspired concentration and as mean ± SEM. Each group contained 9–12 animals. b,c Results at each flow rate were compared independently by ANOVA, which detected a significant effect of exposure concentration (p < 0.01) for both flow rates. Tukey test was then performed; exposure concentration groups with differing superscripts differ at the p < 0.05 level. Open in new tab TABLE 1 URT Uptake of Propylene Oxide . Exposure groupsa . . . . . . 25 ppm . 50 ppm . 100 ppm . 300 ppm . 500 ppm . 12 ml/min 30b,c ± 1.1 33b ± 1.2 29b,c ± 1.4 26c ± 1.0 29b,c ± 0.9 50 ml/min 16b ± 0.7 15b,c ± 1.0 14b,c ± 0.9 11c ± 0.7 12c ± 1.0 . Exposure groupsa . . . . . . 25 ppm . 50 ppm . 100 ppm . 300 ppm . 500 ppm . 12 ml/min 30b,c ± 1.1 33b ± 1.2 29b,c ± 1.4 26c ± 1.0 29b,c ± 0.9 50 ml/min 16b ± 0.7 15b,c ± 1.0 14b,c ± 0.9 11c ± 0.7 12c ± 1.0 a Uptake was measured continuously throughout the 60-min exposure under constant velocity inspiratory flow conditions at the flow rate indicated for the 25-, 50-, 100-, 300-, or 500-ppm exposure groups. Values represent average uptake efficiency during the last 30 min of exposure and are expressed as percentage of the inspired concentration and as mean ± SEM. Each group contained 9–12 animals. b,c Results at each flow rate were compared independently by ANOVA, which detected a significant effect of exposure concentration (p < 0.01) for both flow rates. Tukey test was then performed; exposure concentration groups with differing superscripts differ at the p < 0.05 level. Open in new tab DISCUSSION The current studies revealed both similarities and differences in the URT dosimetry of propylene oxide in the B6C3F1 mouse compared to the F344 rat (Morris et al., 2004). In both species propylene oxide was scrubbed in the URT with moderate to low efficiency (e.g., ≤ 10–33% uptake at inspiratory flow rates approximating one-half to twice the minute ventilation). However, URT uptake efficiencies were statistically different at differing exposure concentrations in the mouse, whereas uptake efficiencies were statistically similar at all exposure concentrations in the rat. Propylene oxide exposure depletes nasal NPSH levels in both species, but the rate of depletion, the maximal degree of depletion, and the exposure concentrations necessary to cause NPSH depletion differed between species. In the mouse, URT uptake demonstrated nonlinear kinetics, with uptake being slightly more efficient at low (in particular, 50 ppm) than at high exposure concentrations (in particular, 300 ppm). This effect was most clear-cut at the higher inspiratory flow rate (50 ml/min), for which uptake efficiency was 15% at 50 ppm compared to 11% at 300 ppm. Since clear-cut concentration-response relationships were not observed, this phenomenon is difficult to precisely interpret. From a total delivered dosage perspective 15 versus 11% represents a relatively narrow range. However, 15% uptake is 1.36-fold higher than 11% uptake, which suggests that differences in local tissue disposition of propylene oxide may exist between low and high exposure concentrations. These results, in particular, the tendency for lower uptake efficiencies at the higher exposure concentrations, may suggest the presence of a saturable uptake pathway. This behavior was not observed for propylene oxide uptake in the rat (Morris et al., 2004). In the rat, uptake averaged 25 and 11% (at inspiratory flow rates approximately one-half to twice the predicted minute ventilation) at exposure concentrations of 25–300 ppm, values very similar to those observed in the mouse at concentrations of 300 and 500 ppm (Table 1). Although some caution should be used in this comparison because the minute ventilation rates as predicted by the equation of Guyton (as described by Phalen, 1984) may not represent precise predictions of the ventilation rates in these species, this comparison does suggest that propylene oxide uptake efficiency at high exposure concentrations is similar in rat and mouse. URT uptake of acetone vapor is similar in urethane-anesthetized rats and mice (Morris, 1991; Morris et al., 1986) as is uptake of styrene vapor in metyrapone-pretreated (to minimize species differences in metabolism) rats and mice (Morris, 2000). Thus, the similarity in uptake between rats and mice at high propylene oxide exposure concentrations is consistent with that of other vapors. In contrast, at low exposure concentrations propylene oxide was scrubbed with greater efficiency in the URT of the mouse than in the rat. Steady-state nasal vapor uptake is dependent on removal of vapor from the nasal tissues via the bloodstream and by reactive pathways (Medinsky et al., 1999; Morris, 1994). Since circulatory removal due to Henry's Law partitioning is a first-order, nonsaturable pathway, it is not likely involved in any zero-order saturable behaviors. Possible saturable pathways include direct reaction with tissue substrates which become depleted at high exposure concentrations or enzymatic pathways with Km values that are low relative to the exposure concentration. A species-specific depletion of a critical propylene oxide reaction substrate in nasal tissues of the mouse but not the rat may explain the differing uptake behavior in these species. However, since NPSH is depleted to a lesser degree in the mouse than rat (see below), it is not likely that NPSH represents such a substrate. Saturation of metabolic pathways may also be responsible for reduced uptake at the higher exposure concentrations. Such behavior has been observed previously for styrene (Morris, 2000) and is perhaps best characterized for acetaldehyde (Morris, 1997; Morris and Blanchard, 1992). Major metabolic pathways for propylene oxide include glutathione-S-transferase and epoxide hydrolase (Faller et al., 2001). It is not thought that metabolic saturation of these pathways occurs in the rat during exposure to concentrations of 500 ppm or less (Morris et al., 2004); however, the Km for propylene oxide metabolism via one or both of these pathways may be lower in the mouse than the rat, leading to saturable behavior. Alternatively, a high-affinity, low-capacity metabolic pathway may be present in the mouse but not rat nasal mucosa. Full metabolic kinetic data on propylene oxide metabolism in nasal tissues of the mouse are not available, precluding any evaluation of this possibility. Since propylene oxide penetrates to the bloodstream (Lee et al., 2005), it is possible that species differences in propylene oxide disposition/metabolism to the bloodstream may also be important in nasal dosimetry of this vapor. Nasal NPSH levels were depleted by propylene oxide in both the mouse and the rat (Morris et al., 2004). In both species the degree of NPSH depletion was not dependent on the inspiratory flow rate despite the fact that delivered dosage rates differed by ∼ 2-fold at the low and high flow rates in both species. As observed for the rat (Morris et al., 2004), this may be due to the intra-animal variability in NPSH content, which was likely sufficiently large to obscure an inspiratory flow rate dependence on NPSH depletion. Both the rate and degree of NPSH depletion during propylene oxide exposure differed between the rat and the mouse. In the mouse exposed to 300 ppm propylene oxide, significant NPSH depletion in both respiratory and olfactory mucosa was observed within 15 min. In the rat 30 min exposure to 300 ppm propylene oxide was required to result in significant depletion. The degree of NPSH depletion is greater in the rat than mouse. This difference is perhaps most evident at an exposure concentration of 100 ppm, which caused significant depletion of respiratory mucosal NPSH in rats (50% of control levels, Morris et al., 2004) but was without effect on NPSH levels in the mouse (100% of control levels, Fig. 3A). Similar relationships were observed in the 300- and 500-ppm exposure groups. Specifically, in the rat, respiratory mucosal NPSH levels averaged approximately 40 and 15% of control levels, respectively, after 1 h exposure to 300 or 500 ppm, compared to 50 and 40% of control levels in mice (Fig. 3A). (The NPSH depletion in olfactory mucosa of the rat was highly variable, making species comparisons difficult.) The kinetic aspects of propylene oxide–induced nasal NPSH depletion are likely complex. In this regard it is important to note that the URT deposition flux rates in this study (e.g., 35–70 nmol/min at 300 ppm) were very high compared to the total amount of NPSH present in control nasal tissues (∼ 80 nmol, respiratory plus olfactory mucosa combined). Thus, the steady-state NPSH levels attained during the 1-h exposures may represent (1) the amount of NPSH in nasal tissue that is sequestered in a site that is unavailable for ready reaction with propylene oxide and/or (2) dynamic steady-state levels that are dependent on the ability of nasal tissues to regenerate NPSH (or glutathione specifically) either from de novo synthesis or via delivery by the bloodstream. Nasal NPSH was depleted to a lesser degree in the mouse than rat. Possible explanations for this observation include a larger pool of NPSH in the mouse that is unavailable for glutathione-S-transferase–mediated reaction with propylene oxide and/or a greater ability of the mouse to rapidly regenerate nasal GSH and maintain NPSH levels (again, either from de novo synthesis or via delivery from the bloodstream). Species differences in total nasal NPSH content are not a likely explanation for the species differences because nasal NPSH levels (expressed per milligram of protein) are similar in the rat and mouse (Lee et al., 2005; Morris et al., 2004). Were rats to have a lesser and/or slower ability to regenerate NPSH, it might provide an explanation for the lower concentration of propylene oxide needed to significantly deplete NPSH in the rat than mouse (100 vs. 300 ppm). A slower regeneration of NPSH in nasal tissues might also provide an explanation for the longer time period needed to reach steady-state NPSH levels during propylene oxide exposure in the rat than in the mouse (30 vs. 15 min, respectively). Future studies are needed to examine these possibilities. In summary, propylene oxide was scrubbed with moderate efficiency (≤ 33%) in the surgically isolated URT of the mouse. URT uptake efficiency demonstrated nonlinear concentration-response relationships with reduced uptake efficiency being observed at 300 ppm compared to 50 ppm exposure concentrations, suggesting that saturation of an uptake pathway, perhaps metabolism, may exist in this species. Nasal respiratory and olfactory mucosal NPSH levels were significantly depleted by 1 h exposure to 300 or 500 ppm propylene oxide. 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Pharmacol. Suppl. 353, R111 . Sedlack, J., and Lindsay, R. H. ( 1968 ). Estimation of total, protein bound, and nonprotein sulfhydryl groups in tissue with Elmann's reagent. Anal. Biochem. 25, 192 –205. Author notes *Toxicology Program, Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269-3092; and †Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674 © The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org TI - Nasal NPSH Depletion and Propylene Oxide Uptake in the Upper Respiratory Tract of the Mouse JF - Toxicological Sciences DO - 10.1093/toxsci/kfj195 DA - 2006-07-01 UR - https://www.deepdyve.com/lp/oxford-university-press/nasal-npsh-depletion-and-propylene-oxide-uptake-in-the-upper-GkoQg0ayTk SP - 228 EP - 234 VL - 92 IS - 1 DP - DeepDyve ER -