Predicted Regional Flux of Hydrogen Sulfide Correlates with Distribution of Nasal Olfactory Lesions in Rats

Frederic J.-M. Moulin,1, Karrie A. Brenneman, Julia S. Kimbell and David C. Dorman,2

CIIT Centers for Health Research, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, North Carolina 27709–2137

Received July 3, 2001; accepted October 17, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Hydrogen sulfide (H2S) is a toxic gas that is released by both natural and industrial sources. H2S selectively targets the olfactory system in humans and rodents. The purpose of this study was to test the hypothesis that the distribution of H2S-induced nasal pathology is correlated with the location of high-flux areas within the upper respiratory tract. To investigate whether the location of the olfactory lesion is dependent on regional gas uptake patterns, a comparison was made between lesion locations and regions of high H2S flux predicted using a 3-dimensional, anatomically accurate computational fluid dynamics (CFD) model of rat nasal passages. Rats were exposed by inhalation to 0, 10, 30, or 80 ppm H2S for 6 h/day for 70 days. The regional incidence of olfactory lesions and predicted H2S flux were determined at the mid-dorsomedial meatus and the middle portion of the ethmoid recess, and their rank correlation was evaluated. At these 2 levels, regions lined by respiratory epithelium were predicted to exhibit the highest mass flux values; however, H2S exposure elicited little or no response in this tissue. In contrast, regions lined by olfactory epithelium showed a close correlation between H2S flux and lesion incidence (p < 0.005) for both the 30 and 80-ppm exposure groups. These results indicate that airflow-driven patterns of H2S uptake within the inherently sensitive olfactory epithelium play an important role in the distribution of H2S-induced lesions and should therefore be taken into consideration when extrapolating from nasal lesions in rats to estimates of risk to human health.

Key Words: hydrogen sulfide; rat; inhalation; pharmacokinetics; computational fluid dynamics; olfactory toxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Hydrogen sulfide (H2S) is a significant environmental contaminant that is produced in nature primarily by the decomposition of organic matter. H2S is also found in natural gas, petroleum, and volcanic and sulfur springs emissions. H2S emissions are associated with more than 70 types of industries, including sewage treatment, swine production, roofing, paper and pulp production, artificial fiber synthesis, and food production (Donham et al., 1982Go; Hall and Rumack, 1997Go; Hoidal et al., 1986Go; Jaakkola et al., 1990Go; Watt et al., 1997Go). However, H2S appears most frequently as an occupational hazard in the petroleum and natural gas industries, which report fatal exposures each year (Arnold et al., 1985Go; Guidotti, 1994Go; Kilburn, 1993Go).

During acute human toxicosis, the observed clinical signs and their severity vary with H2S concentration and duration of exposure. A dose-dependent progression of adverse effects starting with eye irritation at 10–20 ppm and ending with collapse and death at 1000–2000 ppm has been observed following acute H2S exposure (Beauchamp et al., 1984Go; Glass, 1990Go; Reiffenstein et al., 1992Go). Surprisingly, the strong rotten-egg odor of H2S is not a reliable warning sign of its presence since olfactory nerve paralysis rapidly occurs following exposure to >= 100 ppm. With proper medical management, many affected individuals recover fully; however, long-term sequelae of acute H2S poisoning may include organic brain disease resulting from hypoxia as well as hyposmia, dysosmia, phantosmia, and other signs of olfactory dysfunction (Hirsch and Zavala, 1999Go; Kilburn, 1997Go).

Animal studies confirm that the olfactory system is especially sensitive to H2S inhalation. Subchronic exposure of rats to 30 or 80 ppm H2S results in nasal pathology that was limited to the olfactory epithelium (Brenneman et al., 2000Go). Acute exposure of rats to moderately high concentrations of H2S (>= 80 ppm) results in injury followed by regeneration of the respiratory mucosa and full thickness necrosis of the olfactory mucosa (Brenneman et al., in pressGo; Lopez et al., 1988Go). Injury to and regeneration of the respiratory mucosa occurs in animals with ongoing H2S exposure while necrosis of the olfactory mucosa increases in severity (Brenneman et al., in pressGo). These data suggest that the regenerated respiratory epithelium becomes resistant to H2S-induced pathology.

Cytochrome oxidase inhibition is a likely mode of action for H2S-induced olfactory pathology. Decreased cytochrome oxidase activity and increased tissue sulfide concentrations occur in the rat olfactory epithelium following a single 3-h exposure to 80 and 400 ppm H2S, respectively (Dorman et al., 2002Go). Acute inhalation exposure of male rats to 400 ppm H2S also results in severe mitochondrial swelling in degenerating olfactory neurons within the olfactory epithelium (Brenneman et al., in pressGo). This ultrastructural lesion is consistent with H2S-induced anoxic cell injury due to cytochrome oxidase inhibition. These data provide evidence that cytochrome oxidase inhibition may indeed play a critical role in H2S-induced olfactory pathology. These data suggest that the olfactory neuroepithelium is intrinsically more sensitive to H2S-induced nasal pathology than the respiratory epithelium. Regional differences in H2S delivery or uptake and local clearance processes may also contribute to the site-specific distribution of H2S-induced lesions within the rat nose (Morgan and Monticello, 1990Go).

Estimation of chemical delivery to the olfactory region of the rat nose can be difficult. The location of major airflow routes is considered to play an important role in determining olfactory lesion distribution since the air streams flowing through the respiratory tract during breathing influence regional gas uptake (Kimbell et al., 1993Go; Méry et al., 1994Go). Computational fluid dynamics (CFD) models, which are based on anatomically accurate representations of the geometry of the system of interest, provide a useful tool for biologists and toxicologists investigating solute or gas disposition in flow fields (Godo et al., 1995Go, Kimbell et al., 1993Go). CFD models can be used to predict nasal regions with low and high flux during inhalation exposure (Kepler et al., 1998Go; Kimbell et al., 2001Go). Air flow and gas transport are described mathematically within the CFD model by a set of equations describing the laws of conservation of mass and momentum in the inhaled air. The CFD model used in our study has been shown to provide reliable dose estimates of inhaled chemicals in various regions of the rat nose (Bush et al., 1998Go; Hubal et al., 1997Go; Frederick et al., 1998Go).

The purpose of this study was to test the hypothesis that the distribution of H2S-induced nasal pathology is correlated with the location of high-flux areas within the upper respiratory tract. Finding such a correlation would suggest that regional dosimetry is an important contributor to the etiology of the nasal olfactory lesions observed after H2S exposure. Estimates of local tissue dose obtained from similar CFD models of the human nose could then be used to predict H2S exposure concentrations that may result in olfactory toxicity in humans.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Nasal pathology.
The H2S generation and exposure system and nasal histopathology methods have been described in detail previously (Brenneman et al., 2000Go). Briefly, adult male CD rats (n = 12/concentration) were exposed to 0, 10, 30, or 80 ppm H2S, 6 h/day for 70 consecutive days. Rats were euthanized 24 h after the last H2S exposure with CO2 followed by exsanguination. The noses were dissected free and retrograde-flushed with neutral buffered formalin. Transverse nasal sections oriented perpendicular to the bridge of the nose (Fig. 1Go) were obtained at the approximate midpoint of the nasal cavity (section 1) and the middle portion of the ethmoid recess (section 2). These sites were selected because they contain olfactory epithelium and previous studies have shown that these regions are affected by H2S exposure (Brenneman et al., 2000Go). Tissues were stained with hematoxylin and eosin and examined by bright-field light microscopy. Lesion severity in the 2 sections was graded by visual estimation of the percentage of the normal olfactory neuronal cell layer altered by H2S exposure using methods described by Brenneman et al. (2000). Terminology used to describe lesion location was consistent with that defined by Méry and coworkers (1994).



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FIG. 1. (Top) Sagittal view of the rat nose showing normal distribution of the nasal mucosae. The black diagonal lines indicate the 2 section levels examined. (Middle) Schematic diagrams of the 2 transverse nasal sections evaluated. DMM, dorsal medial meatus; ET, ethmoid turbinate; LD, nasolacrymal duct; IN, incisor. (Bottom) Numbers indicate regions of olfactory epithelium evaluated.

 
Using maps of rat nasal transverse sections, the olfactory-lined portion of the airway perimeter was divided into regions of roughly similar size (ranging from 300 to 1000 µm in length) based on anatomical landmarks. Four and 39 olfactory regions were demarcated in nasal sections 1 and 2, respectively (Fig. 1Go). On the right hemisection, the regions were numbered consecutively along the nasal cavity perimeter beginning with region 1, which corresponds to the roof of the dorsal medial meatus (Fig. 1Go). The presence or absence of olfactory lesions that were at least moderately severe was determined in all animals for each region in both sections. A region was considered affected by H2S if 50% or more of the region had at least a moderate-grade lesion. Changes in epithelia found with similar frequency and severity in control and exposed groups were regarded as background findings and excluded from the analysis. The incidence of olfactory lesions was calculated for each region of both nasal sections.

Computer simulations.
Inspiratory airflow and H2S uptake were simulated using a modified version of the CFD model previously described by Kimbell et al. (1997a,b) for formaldehyde. This model consists of a 3-dimensional reconstruction of serial cross sections obtained from an adult Fischer-344 male rat nose (Fig. 2AGo). The set of equations used to simulate airflow and mass transport was solved by a commercial software package (FIDAP, Fluent Inc., Lebanon, NH) using the finite element method. In this method, the volume of the nasal cavity is divided into a series of simple 3-dimensional compartments, and the equations are solved separately in each element. The elements are then reassembled to provide a simulation of the flow and mass transport through the entire complex airspace.



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FIG. 2. (A) Computational mesh generated by FIDAP representing the rat nasal cavity and viewed from the septal side. Nostril is to the left and the nasopharyngeal duct to the right. (B) Location of computer-generated cross sections within the mesh corresponding to nasal sections 1 and 2.

 
Hydrogen sulfide flow simulations were conducted at steady state in the inspiratory direction. Airflow velocity was set to a constant value perpendicular to the nostril surface plug flow and to 0 at the nasal walls. The minute volume used for model simulations was based on an average 20-week-old male rat and was estimated to be 288 ml/min (Kimbell et al., 1997aGo). We also assumed that exposure to H2S did not affect either the density or viscosity of the inspired air or the shape of the nasal cavity. Transport of H2S through the air and into the nasal epithelium was assumed to occur by convective forces and molecular diffusion (see Appendix for details).

In order to solve the transport equation for H2S in the nasal airway lumen, information on H2S uptake at the air/nasal tissue interface was needed. This information was implemented in the CFD simulations through the specification of conditions on uptake at the boundaries or walls of the lumen. Boundary conditions can be implemented by specifying H2S concentration, flux, or flux as a function of concentration. There was not enough information on the solubility and reactivity of H2S in the nasal passages to develop a boundary condition in which flux is a nonlinear function of concentration. A concentration boundary condition where concentration is set to nonzero values was not realistic here since nasal tissue is not currently known to maintain constant levels of H2S. A zero-valued constant concentration boundary condition mimics the most efficient uptake scenario in which 100% of inhaled H2S is extracted by nasal tissues upon inspiration. The use of this condition would be justified if H2S were extremely water-soluble. Since H2S has been described as highly water-soluble (Lopez et al., 1988Go) as well as very water-insoluble (Witschi and Last, 1996Go), we used boundary conditions in which flux was a linear function of concentration to mimic the more moderate uptake scenarios of 20, 40, and 80% extraction of H2S by nasal tissues to model low, medium, and high uptake, respectively. No attempt was made to separate water solubility from other factors that may affect H2S uptake by nasal tissues. The constants of proportionality between nasal wall flux and concentration used in boundary conditions in these simulations represented mass transfer coefficients that group or lump the effects of solubility, reactivity, tissue morphometry, and clearance mechanisms together.

Finally, all nasal walls were assumed to have the same ability and capacity to absorb H2S regardless of anatomical location or lining epithelium. Regional variation in H2S flux at the nasal walls was therefore due primarily to regional distribution via airflow inspiratory patterns.

Nasal cross sections were identified on the computer 3-dimensional model of the nose corresponding to the 2 sections used for the nasal pathology (Fig. 2BGo). The perimeters of these sections were divided into regions following anatomical landmarks identical to those used to record lesion incidence. The net rate of transfer (flux) to nasal walls was modeled. The fluxes simulated in all the different elements included within a section were averaged (see Appendix). All the regions from each section were ranked by average flux and by lesion incidence. Correlations between rankings of simulated flux and lesion incidence were determined using Spearman's rank correlation coefficient (Mendenhall et al., 1981Go). A p value < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Incidence of Olfactory Lesions in H2S-Exposed Rats
Following exposure to 30 and 80 ppm H2S, a statistically significant increase in nasal lesions limited to the olfactory mucosa was observed (Brenneman et al., 2000Go). The lesions consisted of bilaterally symmetrical olfactory neuronal loss and basal cell hyperplasia that occurs in discrete regions within the olfactory epithelium (Fig. 3Go). The composite distribution of nasal lesions resulting from subchronic H2S exposure has been described in detail elsewhere (Brenneman et al., 2000Go). In section 1, pathology was limited to the olfactory mucosa lining the dorsal medial meatus. In section 2, H2S-related lesions were only observed at specific sites within the olfactory epithelium even though the olfactory epithelium is broadly distributed. These sites included the nasal septum, dorsal walls of the nasal cavity, and margins of the ethmoturbinates. The incidence, severity, and distribution of exposure-related lesions increased with concentration, affecting approximately 50% of the olfactory epithelium at 30 ppm and 70% at 80 ppm (Tables 1 and 2GoGo). Rats exposed to 10 ppm were unaffected.



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FIG. 3. Photomicrograph of the olfactory mucosa-lined ethmoturbinate (ET) in the nasal cavity demonstrating the presence of olfactory neuronal loss within discrete regions of the rat nose (arrows and box). Inset demonstrates a higher-magnification photomicrograph of the affected region outlined with a box in the main figure. NS, nasal septum.

 

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TABLE 1 Regional Nasal Lesion Incidence and Predicted H2S Flux for Section 1
 

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TABLE 2 Regional Nasal Lesion Incidence and Predicted H2S Flux for Section 2
 
Regional H2S Nasal Dosimetry as Predicted by CFD Modeling
For section 1, high olfactory H2S fluxes were predicted to occur in the dorsal medial meatus on the dorsal septum and the adjacent lateral wall (Fig. 4Go). For all regions of the dorsal medial meatus, simulations assuming high H2S uptake predicted flux values higher than those obtained from simulations assuming medium uptake, which were higher than simulations based on low uptake (Table 1Go).



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FIG. 4. Schematic diagram of the transverse nasal section evaluated (left) and plots of lesion incidence at 30 and 80 ppm (top) and predicted H2S flux (pg/min2-s) at 80 ppm (bottom) on designated regions of section 1.

 
For section 2, high olfactory H2S fluxes were predicted to occur on the septum and medial aspects of the first, third, and fifth ethmoturbinates (Fig. 5Go). Peak flux values predicted by the low, medium, and high uptake simulations in these regions were within 3-fold of one another, with the highest peak flux values predicted by the medium uptake simulation. Flux values calculated for an 80-ppm exposure are presented in Table 2Go for the 3 uptake scenarios. In all the simulations, flux at a given site was a linear function of inhaled H2S concentration. Thus, estimated flux values for 30 ppm were derived from the 80-ppm values by multiplying by 30/80. Error in the mass balance of simulated H2S transport was less than 10% for all simulations.



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FIG. 5. Schematic diagram of the transverse nasal section evaluated (top left), plot of lesion incidence at 30 and 80 ppm (top right), predicted H2S flux (pg/(mm2-s)) at section 2 under intermediate uptake conditions at 80 ppm where red corresponds to 320 pg/(mm2-s) and blue corresponds to 0 pg/(mm2-s) (bottom left), and plot of predicted flux at 80 ppm (bottom right) on designated regions of section 2.

 
Comparison of Flux Predictions and Observed Olfactory Lesions
On both sections, regions were ranked by flux and lesion incidence. In section 1, only the olfactory epithelium lining the dorsal medial meatus was affected by H2S exposure, and yet the walls of the medial meatus lined by respiratory epithelium was the area of section 1 where the highest flux of H2S was predicted. Similarly, in section 2, the respiratory epithelial lining of the ventral meatus showed a large predicted transfer of H2S and yet was unaffected by H2S exposure.

When the comparison between lesion incidence and regional flux was restricted to areas solely lined by olfactory mucosa, there was a marginal correlation on section 1 and a significant correlation on section 2 (Figs. 4 and 5GoGo; Table 3Go). Despite overall correlations, discrete regions demonstrated discrepancies between estimated flux and nasal lesions. For example, at 80 ppm, the first and second ethmoturbinates (regions 5–12, Fig. 1Go) had pathology despite the estimation of very low flux. In contrast, the ventral margins of the third ethmoturbinate and the lateral aspect of the fifth ethmoturbinate at section 2 (regions 24–30, Fig. 1Go) showed relatively high levels of flux with no associated nasal lesion.


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TABLE 3 Correlation between Observed Olfactory Lesion Incidence Induced by H2S and Predicted H2S Flux Calculated Using a 20, 40, or 80% Extraction Coefficient
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
The olfactory epithelium is clearly sensitive to H2S, and this research was intended to address whether regional dosimetry within this site could be a determinant of toxicity. A variety of experimental approaches can be used to estimate the amount of chemical delivered to the different nasal mucosae following inhalation exposure. Direct measurement of sulfide content in the nasal epithelium following H2S exposure has been attempted but is difficult due to high endogenous sulfide levels and the small size of rat nasal mucosal samples (Dorman et al., 2002Go). Our laboratory has found that endogenous sulfide levels in the rat olfactory and respiratory nasal mucosa are approximately 2–2.5 ppm. We have also shown that increased olfactory epithelial concentration occurs in rats immediately following the end of a 3-h, nose-only exposure to 400 ppm H2S (Dorman et al., 2002Go). Although not statistically significant, qualitatively similar increases in respiratory epithelial sulfide concentration also occurred. These studies relied on analysis of the entire mucosal sample and therefore cannot provide accurate estimates of delivered tissue dose within more discrete regions of the rat nose.

The use of CFD models provides an attractive alternative for estimating regional delivery of inhaled H2S to the rat nose. Correlation of formaldehyde flux predictions with the location of formaldehyde-induced nasal lesions was previously demonstrated using this CFD model of the Fisher 344 rat nose (Kimbell et al., 1997bGo). Kimbell and coworkers (1997a) estimated that approximately half the total volume of inspired air follows 3 major pathways, namely the dorsal medial meatus, the middle meatus, and the ventral medial meatus. The dorsal medial air stream is the main route delivering inhaled chemicals to the olfactory-lined regions of the nasal cavity (Kimbell et al., 1997aGo).

Analysis of predicted H2S concentrations in the 2 nasal sections examined in our study showed that the air streams travelling through these 3 passages contained high concentrations of H2S. Since high airspeed is also found in these air streams, we expected that a large amount of H2S would be absorbed by the nasal epithelium bordering these air streams. This was indeed the case for model predictions for both sections. Distinct hot spots of regional flux occurred at the level of section 1 along the middle and dorsal medial meati and at the level of section 2 in the dorsal medial region of the ethmoid recess, the middle and ventral meati, and the nasopharyngeal duct. These regions of high flux were also associated with H2S-induced nasal lesions if the airway was lined by olfactory epithelium. Inhalation of other direct irritant gases such as chlorine and dimethylamine result in similar olfactory lesions at these sites (Buckley et al., 1985Go; Jiang et al., 1983Go; Morgan et al., 1991Go).

Previous CFD simulations have predicted that the location of major nasal airflow streams in the inspiratory direction do not change significantly with inspiratory airflow rate (Kimbell et al., 1997aGo). There is little information available about the potential effects of inlet velocity profiles on downstream (posterior) airflow patterns. However, flow patterns predicted by simulations in which a constant-velocity condition (plug flow) was used at the nostril compared well with dye-streakline visualizations of flow where nostril velocity profiles were presumably not constant (Kimbell et al., 1993Go, 1997aGo). Therefore effects of different inlet velocity profiles on predicted airflow and H2S flux patterns posterior to the nasal vestibule were assumed to be insignificant.

Unlike what would have been expected if H2S were a direct irritant, areas with predicted high H2S flux did not always correlate with nasal pathology. We did not observe respiratory epithelial pathology regardless of H2S exposure concentration, anatomical location, or predicted flux. For example, the respiratory epithelium-lined lateral walls and nasopharyngeal duct appear to be high flux areas. However, these regions did not develop pathology following subchronic H2S exposure. Acute (3-h) H2S exposures of rats to >= 80 ppm H2S induced respiratory epithelial regeneration on the lateral wall of the ventral meatus (Brenneman et al., in pressGo). However, this pathology was not observed following 5 consecutive days of H2S exposure to 80, 200, or 400 ppm (Brenneman et al., 2000Go). The transient nature of respiratory epithelium sensitivity to H2S suggests that rapid tissue regeneration or metabolic adaptation of the respiratory epithelium may occur following acute H2S exposure. Adaptive regeneration of the respiratory epithelium to inhaled irritants such as ozone has been described previously (Harkema et al., 1997Go). However, this effect usually involves metaplastic modifications of the epithelium, an element absent from the response to repeated H2S exposure (Brenneman et al., in pressGo).

In view of the relative resistance of the respiratory mucosa to H2S toxicity, we focused on the relationship between regional flux predictions and olfactory epithelial damage. At the level of section 1, the correlation between H2S flux and lesion development was only marginal. One possible explanation is that the very small amount of olfactory tissue present at that level, limited to the lining of the dorsal-medial meatus, did not provide an adequate number of regions for comparison. In contrast, section 2, with most of the surface lined by olfactory epithelium, showed a significant correlation between areas of high flux and location of olfactory lesions. This correlation between H2S flux and lesion incidence supports the hypothesis that incidence and severity of lesions in the olfactory mucosa are related to the amount of H2S reaching the tissues.

A few discrepancies between predicted flux and tissue response were noted in the more posterior section. These discrepancies could arise from either site-specific uptake factors in the exposed animals or CFD model inaccuracies. On the first and second ethmoturbinates, where lesions occurred but low flux values were predicted, tissue effects may result from a culmination of repeated exposure by each breath, whereas the simulation only predicts flux from inspiratory airflow at steady-state. On the ventral scroll of the third ethmoturbinate and the lateral aspect of the fifth ethmoturbinate, where flux was predicted to be high but very little pathology was observed, there might be site-specific metabolic or clearance factors that affect the disposition of H2S within the tissue. Such discrepancies also highlight the need for further confirmation studies of CFD models.

Precise information concerning the relative solubility of H2S gas in water or lipids is lacking. Therefore CFD model simulations were performed without accounting for differences in H2S uptake by dry tissues lining the nasal vestibule and mucus-coated tissues lining the rest of the nasal passages. In addition, since the amount or percentage of inhaled H2S retained by the rat nasal passages during inspiration is unknown, simulations were conducted under conditions representing low, medium, and high water uptake. These simulations, as well as a simulation using zero-valued constant concentration (C = 0) at nasal walls showed very similar profiles of flux throughout the regions. Results from all simulations correlated with the location of nasal lesions, with the exception of C = 0 flux at the more anterior sections, suggesting that nasal uptake patterns are relatively insensitive to overall uptake efficiency.

The most significant difference among the 3 simulations was the size of predicted H2S flux. In general, the medium uptake simulations predicted the largest flux to damaged areas. This finding was due to the combination of the location of section 2 and the extraction rates used. Section 2 was located near the middle of the olfactory area, near the posterior aspect of the middle regions of the nasal passages. When overall uptake was high most of the extraction of inhaled H2S was predicted to occur in the anterior nose, leaving less gas to contribute to flux in the middle and posterior parts of the nose. When uptake was low, lower H2S extraction was predicted to occur in the anterior and middle portions, leaving more gas to contribute to flux in the posterior nose. Because of this pattern, fluxes estimated in the middle region of the nose would be expected to be highest for the intermediate uptake case. These observations suggest that an intermediate uptake rate would result in the largest olfactory tissue exposure. Our results also imply that water solubility alone is unlikely to represent a critical element for the pathogenesis of H2S-induced olfactory lesions, a result that is inconsistent with a potential mechanism of toxicity resulting from simple direct contact of a reactive product. Current predictions of H2S flux from each simulation using different uptake conditions should be considered relative and quantitative values used only to set bounds on the probable range of regional mass transfer. Comparisons of flux values predicted in the rat to those in another species could be conducted for identical uptake conditions but would remain speculative until more refined estimates of nasal extraction efficiency become available.

Overall, the results presented here suggest that H2S nasal toxicity depends on tissue sensitivity and delivered dose within the sensitive tissue. With absorption characteristics of nasal walls assumed to be similar throughout the nose, higher H2S flux was predicted in regions lined by respiratory epithelium due to their more anterior and ventral locations. Since H2S primarily affects the olfactory epithelium, sparing the respiratory mucosa, olfactory cells are evidently more susceptible to toxicity regardless of regional dosimetry. Further research is required to evaluate the difference in distribution of H2S-sensitive cellular enzymes between the respiratory epithelium and olfactory mucosa to understand the basis of their different sensitivities. The results of our study indicate that airflow-driven patterns of uptake play an important role in the distribution of H2S-induced olfactory lesions in the rat. Thus, species-specific regional airflow in the nasal cavity should be taken into account when estimating the risk presented to humans by chronic H2S exposure.


    APPENDIX
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
A description of the governing equations and boundary conditions for the CFD simulations presented here is similar to the description given by Hubal et al. (1996) for simulation of the uptake of inhaled ozone in the anterior rat nasal passages. The governing equations for airflow and hydrogen sulfide transport are the Navier-Stokes equations representing conservation of momentum (Equation 1Go), and the continuity (Equation 2Go) and convective-diffusion (Equation 3Go) equations representing conservation of mass:


((1))

((2))

((3))
where u, {rho}, and µ are the air velocity, density, and viscosity, respectively; p is the pressure; C is the concentration of inhaled hydrogen sulfide; Da is the diffusivity of hydrogen sulfide in air (0.15 cm2/s, estimated using a formula given by Hobler [1966]); and {triangledown} and {triangledown}2 are the gradient and Laplace operators, respectively.

At 30 and 80 ppm hydrogen sulfide is very dilute, so we assumed the gas did not significantly affect the density or viscosity of air. Thus hydrogen sulfide had no effect on airflow and Equations 1 and 2 GoGocould be decoupled from Equation 3Go. This meant that Equations 1 and 2 GoGocould be solved first and the resulting flow field (u) used as input to Equation 3Go.

To solve these equations, boundary conditions must be prescribed at the nostril and outlet surfaces and at nasal walls. For airflow, a constant velocity profile was imposed perpendicular to element faces making up the surface of the nostril (plug flow). Air velocity was set to 0 at nasal walls (no-slip condition). A stress-free condition, arising naturally from the finite element method that was used to discretize Equations 1, 2, and 3GoGoGo, was imposed at the outlet. This condition forces the normal stress to be equal to 0 in a weighted sense.

To calculate an airflow rate, the amount of inspired air (tidal volume, VT) was divided by the estimated time involved in inhalation (half the time a breath takes, or (1/2)(1/(breathing frequency, f)). Thus an inspiratory flow rate was calculated to be 2VTf or twice the minute volume. The minute volume for a 315 g rat (as was used to construct the CFD model) was allometrically scaled to 0.288 l/min from data given by Mauderly (1986). Thus airflow simulation for 1 nostril was carried out at a Reynolds number (Re) equal to 243 (Re = {rho}LU/µ, {rho} = density of air, 1.2 x 10–3 g/cm3; µ = viscosity of air, 1.8 x 10-4 g/(cm-s)) corresponding to a volumetric flow rate for 2 nostrils of 0.576 l/min. Airflow simulations compared well with descriptions and measurements of flow in nasal molds (Kimbell et al., 1993Go, 1997aGo).

For solution of Equation 3Go, the concentration of inhaled hydrogen sulfide was specified at the nostril and a linear relationship between flux and concentration (boundary condition of the third kind) was applied at the nasal walls (Equation 4Go):


((4))
where Nwall is the wall mass flux of hydrogen sulfide, Cwall is the hydrogen sulfide concentration on the air side of the air-tissue interface, and h is a tissue-side mass transfer coefficient that was fitted so that desired values were obtained for the total nasal extraction of hydrogen sulfide. At the outlet, the concentration gradient was set equal to 0. Hydrogen sulfide mass transport simulations were conducted at a Peclet number (Pe) equal to 243 (Pe = LU/Da, L = hydraulic diameter of nostril, 0.1136 cm; U = characteristic speed, 321 cm/s; Da = diffusivity of hydrogen sulfide in air, 0.15 cm2/s as above).

To estimate flux at the boundary during post-processing of completed simulations, the following formula was used:


where N is the flux, xj refers to the jth coordinate direction, nj is the jth component of the surface normal vector, ui is the ith component of velocity, and the summation convention is assumed. This formula was implemented by the postprocessing module (FIPOST) of the FIDAP software package (Fluent, Inc., Lebanon, NH) used to conduct the airflow and hydrogen sulfide uptake simulations. Fluxes simulated in different elements included within a single region were averaged without area weighting since mesh elements were relatively uniform in size.


    ACKNOWLEDGMENTS
 
This study was funded in part by a grant from the American Petroleum Institute (API). The authors acknowledge the technical assistance of the members of the CIIT animal care, inhalation, and pathology service groups. We appreciate the comments of Drs. Paul Foster, Fred Miller, and Barbara Kuyper during their review of the manuscript.


    NOTES
 
1 Present address: Department of Lead Safety Assessment, Bristol-Meyers Squibb Pharmaceutical Research Institute, Hopewell, NJ 08543–4000 Back

2 To whom correspondence should be addressed. Fax: (919) 558-1300. E-mail: dorman{at}ciit.org. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Arnold, I. M., Dufresne, R. M., Alleyne, B. C., and Stuart, P. J. (1985). Health implication of occupational exposures to hydrogen sulfide. J. Occup. Med. 27, 373–376.[ISI][Medline]

Beauchamp, R. O., Jr., Bus, J. S., Popp, J. A., Boreiko, C. J., and Andjelkovich, D. A. (1984). A critical review of the literature on hydrogen sulfide toxicity. Crit. Rev. Toxicol. 13, 25–97.[Medline]

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