Lowest observed adverse effect level of pulmonary pathological alterations due to nitrous acid exposure in guinea pigs

Male Hartley guinea pigs (n = 20; body weight, approximately 360 g; age, 6 weeks) were purchased from SLC, Japan (Shizuoka, Japan). The animals were divided into four groups (n =5/group) and preliminarily housed for 1 week in individual animal-exposure chamber with filtered room air [10]. Briefly, the animals were raised in 0.2 m³ hand-made acrylic chambers with a 16 L/min flow volume of filtrated room air, using about 5 kg charcoal activated granular, 15 sheets of American air filters for vinyl isolator (Clea Japan, Inc., Tokyo, Japan), air compressors (0.4LE-8S; Hitachi Industrial Equipment Systems Co., Ltd., Tokyo, Japan), dehumidifiers (RAX3F; Orion Machinery Co., Ltd., Nagano, Japan), high-pressure regulator valves (with the largest supply pressure of about 0.078 MPa; model no. 44–2263-241; Kojima Instruments Inc., Kyoto, Japan) and mass flow controllers to control air flow (model 8350MC-0-1-1; Kojima Instruments Inc.). The internal pressure in the chambers was adjusted to about + 1 mm H₂O relative to atmospheric pressure. Food and water were available freely during all experimental periods. The animal room was maintained under a dynamic temperature of 25 ± 2 °C to stabilize the chamber temperature and humidity. The room lighting was turned on/off by staff at 9:00 and 17:30.

In the animal-exposure chambers, the guinea pigs were exposed to filtered room air (control group: C group) or filtered room air after passing through three HONO-generation systems (low-, middle-, and high-concentration groups: L group, M group, and H group) [17]. Briefly, the HONO-generation system was based on spraying a mixture of aqueous sodium nitrite solution (> 98.5% pure sodium nitrite; Wako Pure Chemical Industries, Ltd.) and aqueous acid solution (85–92% lactic acid; Wako Pure Chemical Industries, Ltd.) in a porous polytetrafluoroethylene tube (TB-1008, approximately 15 cm length; Sumitomo Electric Fine Polymer, INC., Osaka, Japan) using filtered room air through an atomizer-nozzle (BN90s-IS[V], SUS316L, 1/8PT, M14; Atomax Co., Shizuoka, Japan). The filtered room air passing through three HONO-generation systems flowed into three HONO-exposure chambers. The aqueous solution inside the tube of HONO generation systems was discharged with a small portion of the gas. The HONO concentrations in the animal-exposure chambers were regulated based on the concentration of the aqueous sodium nitrite solution (L, M, and H groups: 2, 6, and 18 mmol/L). Guinea pigs were continuously exposed to HONO for 4 weeks. HONO exposure was stopped for approximately 3 h once every week to allow for the exchange of cages and cleaning of chambers.

The HONO-measurement system has been described previously [10]. Briefly, we used a sampling method of HONO, employing two Harvard EPA Annular Denuders (URG-2000-30 × 150-3CSS; URG Corporation, NC) in the series sampling [18, 19]. The annular denuders were coated with sodium carbonate and glycerol. Air from each chamber was sampled for 30 min per day for 5 days per week with an air flow of 1 L/min, using NOx analyzer. The concentration of HONO in the air of each chamber was measured after the end of the exposure experiment, using the denuder extract with milliQ by ion chromatography (700 series; Metrohm Japan LTD., Tokyo, Japan).

The concentrations of the contaminated NO₂ and NO were measured by a NOx analyzer (ECL-77A; J-science, Inc., Kyoto, Japan) after passing into the sodium carbonate annular denuders.

All animals were sacrificed after 4 weeks of HONO exposure using an overdose of pentobarbital sodium and exsanguination. For light microscopy, lung tissue samples of three guinea pigs were fixed with 10% neutral-buffered formalin (Mildform 10 NM; Wako Pure Chemical Industries, Ltd.) at 20 cm/H₂O for the alveolar distension in each group. The lung samples were embedded in paraffin. Tissues were then sectioned and deparaffinized, stained with hematoxylin and eosin (HE) and Elastica van Gieson (EVG), and examined under a light microscope. The Lm, a measure of airspace enlargement, was examined [12]. Using light microscopy images, Lm was determined using the transparent overlay tracing of a 0.1-mm mesh hemocytometer. All intercepts with alveolar septal walls were counted at the intersection point of around five non-adjacent meshes that did not intercept bronchus or blood vessels. The total length (2 mm) of all the lines combined divided by the total number of intercepts yielded the Lm for the region studied [20, 21]. The thickness of the bronchial smooth muscle layer near the middle of the bronchus was measured using right-lung middle lobe samples. Using light microscopy, the thickness of the bronchial smooth muscle layer of each guinea pig was determined at five points around one bronchus [12].

For transmission electron microscopy (TEM) and scanning electron microscopy (SEM), lung tissue samples of two guinea pigs were fixed with 1% paraformaldehyde electron microscopy grade (TAAB Laboratories Equipment, Ltd., Berkshire, England) and 1% glutaraldehyde (20% glutaraldehyde solution; Wako Pure Chemical Industries, Ltd.) phosphate buffer at 20 cm/H₂O for the alveolar distension in each group. The tissues were treated by routine methods and examined under TEM (JEM-1200 EX; JEOL Ltd., Tokyo, Japan) and SEM (JSM-T100; JEOL Ltd).

Table 1 shows the concentrations of HONO and secondary products of NO₂ and NO in the animal-exposure chambers. The total sampling periods were < 2% throughout the experimental period. Although secondary products of NO were > 0.1 ppm in the M and H groups, secondary products of NO₂ were < 0.1 ppm.

Figure 1 shows the body weights of guinea pigs in each group. Although a tendency for dose-dependent decrease in body weight due to HONO exposure was observed, no significant differences were found by ANOVA.

Figure 2a shows the main bronchus near the middle of the right-lung middle lobe of guinea pigs in each group. The bronchoconstriction varied in each animal, and thus a comparison between the groups was not clear. The boundary of the smooth muscle layer in the bronchial connective tissue was clear in each group. The average thickness of the bronchial smooth muscle layer near the middle of the right-lung middle lobe is shown in Table 2. Although a tendency for thickening of bronchial smooth muscle due to HONO exposure was observed, there was no significant difference. Figure 2b shows the magnified images of Fig. 2a. There were no inflammatory alterations in any group. However, in the H group, a tendency for hyperplasia and pseudostratification of bronchial epithelial cells was observed. Figure 2c shows the alveolar duct regions of guinea pigs in each group. A dose-dependent extension of bronchial epithelial cells in the alveolar duct regions was observed, with the smooth muscle cells.

Figure 3a shows the alveolar duct regions of guinea pigs in each group. Hypertrophic alterations of the cells were observed in the alveolar duct interstitial regions in guinea pigs of the H group. A flattened surface (without fibrosis) was observed in the alveolar duct regions, and this was found to be dose-dependent. Figure 3b shows the alveoli of the guinea pigs in each group. Dose-dependent pulmonary emphysema-like alterations in the alveolar duct centriacinar regions were observed in guinea pigs of the HONO-exposure groups. Morphometric quantification of the extent of emphysema-like alterations is shown in Table 3. The average Lm of each group was 39.8–59.4 mm. The Lm of the H group was significantly longer than that of the C group according to Dunnett’s multiple comparison, but not ANOVA. Figure 3c shows the TEM images (× 5000 magnification) of the interstitium of the alveolar duct regions in guinea pigs of each group. Smooth muscle cells with abundant mitochondria and intermediate microfilaments were observed in the interstitium of the alveolar duct regions in the L, M, and H groups (consistent with the observations in Fig. 2c). The smooth muscle cells which in the interstitium of the alveolar duct regions with abundant mitochondria and intermediate microfilaments, consistent the observations in Fig. 2c. There were no edematous alterations of guinea pigs in any group.

Figure 4a shows the SEM images (× 500 magnification) of bronchial lumen near the main bronchus in guinea pigs of each group. The cilia of bronchial epithelial cells in guinea pigs from all the HONO-exposure groups showed no damage. A flattened surface of the bronchial epithelial cells was observed in the bronchial lumen of guinea pigs from the M and H groups, and this change was dose-dependent. Figure 4b illustrates the SEM images (at × 100 and × 200 magnifications) of the terminal bronchiole regions of guinea pigs in each group. A dose-dependent proliferative alteration of the cells was observed in the alveolar duct regions. Figure 4c demonstrates the alveolar regions of each group at × 200 magnification. Although the peripheral alveoli in the C group were normally inflated, the peripheral alveoli in the HONO-exposure groups (L, M, and H groups) were pulled in all directions and had small inflations, and this effect was dose-dependent. While the peripheral alveoli of the C group appeared to expand with low tension, the peripheral alveoli of the HONO-exposure groups (L, M, and H groups) appeared to expand with high tension. In contrast, the alveolar duct lumen of the HONO-exposure groups showed a dose-dependent expansion.