Elizabeth A. Gross

Modeling interindividual variation in physiological factors used in PBPK models of humans.

Modeling interindividual variation in internal doses in humans using PBPK models requires data on the variation in physiological parameters across the population of interest. These data should also reflect the correlations between the values of the various parameters in a person. In this project, we develop a source of data for human physiological parameters where (1) the parameter values for an individual are correlated with one another, and (2) values of parameters capture interindividual variation in populations of a specific gender, race, and age range. The parameters investigated in this project include: (1) volumes of selected organs and tissues; (2) blood flows for the organs and tissues; and (3) the total cardiac output under resting conditions and average daily inhalation rate. These parameters are expressed as records of correlated values for the approximately 30,000 individuals evaluated in the NHANES III survey. A computer program, Physiological Parameters for PBPK Modeling (P3M), is developed that allows records to be retrieved randomly from the database with specification of constraints on age, sex, and ethnicity. P3M is publicly available. The database and accompanying software provide a convenient tool for parameterizating models of interindividual variation in human pharmacokinetics.

Nasal Diagrams: A Tool for Recording the Distribution of Nasal Lesions in Rats and Mice:

Knowledge of patterns of lesion distribution can provide insight into the relative roles played by regional tissue dose and local tissue susceptibility in toxic responses to xenobiotics in the nose and assist assessment of potential human risk. A consistent approach is needed for recording lesion distribution patterns in the complex nasal airways of rats and mice. The present work provides a series of diagrams of the nasal passages of the Fischer-344 rat and B6C3F1 mouse, designed for mapping nasal lesions. The diagrams present each of the major cross-sectional airway profiles, provide adequate space for nasal mucosal lesion recording, and are suitable for duplication in a commercial photocopier. Sagittal diagrams are also provided to permit transfer oflesion location data observed in transverse sections onto the long axis of the nose. The distribution of lesions induced by a selected range of xenobiotics is presented. Approaches to application of the diagrams and interpretation of results obtained are discussed in relation to factors responsible for lesion distribution in the nose and their relevance to interspecies extrapolation. A modified approach to anatomical classification of the ethmoturbinates of the rodent is also presented.

Olfactory Neuron Loss in Adult Male CD Rats Following Subchronic Inhalation Exposure to Hydrogen Sulfide

Dysosmia and anosmia are reported to occur following human exposure to hydrogen sulfide (H2S) gas. The clinical association between H2S exposure and olfactory dysfunction in humans necessitates evaluation of the nasal cavity and olfactory system in experimental animals used to study H2S toxicity. The purpose of this study was to subchronically expose 10-week-old male CD rats to relatively low concentrations of H2S and to histologically evaluate the nasal cavity for exposure-related lesions. Rats (n = 12/ group) were exposed via inhalation to 0, 10, 30, or 80 ppm H2S 6 h/d and 7 d/wk for 10 weeks. Following exposure to 30 and 80 ppm H2S, a significant increase in nasal lesions limited to the olfactory mucosa was observed. The lesions, which consisted of olfactory neuron loss and basal cell hyperplasia, were multifocal, bilaterally symmetrical, and had a characteristic rostrocaudal distribution pattern. Regions of the nasal cavity affected included the dorsal medial meatus and the dorsal and medial portions of the ethmoid recess. The no observed adverse effect level for olfactory lesions in this study was 10 ppm. For perspective, the American Conference of Governmental Industrial Hygienists threshold limit value (TLV) recommendation for H 2S is currently 10 ppm (proposed revision: 5 ppm), so the concentrations employed in the present study were 3 and 8 times the TLV. These findings suggest that subchronic inhalation exposure to a relatively low level of H2 S (30 ppm) can result in olfactory toxicity in rats. However, because of differences in the breathing style and nasal anatomy of rats and humans, additional research is required to determine the significance of these results for human health risk assessment.

Relationship between benzene toxicity and the disposition of 14C-labelled benzene metabolites in the rat

The distribution of radioactivity associated with three 14C-labelled benzene metabolites was studied using whole body autoradiography (WBAR). Male Fischer-344 rats were given an intravenous dose of 0.6 mg/kg (60 microCi phenol, 1.2 mg/kg (100 microCi) catechol, or 1.3 mg/kg (100 microCi) hydroquinone. The rats were killed after 2 h and autoradiograms were prepared from whole body sagittal sections. The relative organ uptake of radioactivity associated with each compound was assessed by comparing tissue/blood optical density (O.D.) ratios from X-ray films. Bone marrow, thymus and the white pulp of the spleen concentrated radioactivity associated with hydroquinone or catechol. Radioactivity associated with phenol concentrated in the red pulp of the spleen, but not in the other lymphoid tissues. Radioactivity associated with all three metabolites was found in the lungs, kidneys and small intestines, whereas greater accumulation of radioactivity was observed in subcutaneous tissues, sebaceous glands and the white matter of the brain and spinal cord in rats given hydroquinone or catechol than in animals given phenol. Rats pretreated with a single dose of Aroclor 1254 (250 mg/kg, i.p.), a regimen which was found to protect against benzene-induced lymphocytopenia, were given hydroquinone (100 microCi; 1.3 mg/kg) or catechol (100 microCi; 1.4 mg/kg). For hydroquinone the tissue/blood O.D. ratios for bone marrow and thymus were approx. 60% lower in Aroclor-pretreated than in untreated rats. A 25% reduction in the tissue/blood O.D. ratios for these organs was observed in pretreated rats given catechol. These findings indicate that the uptake and concentration of radioactivity associated with hydroquinone and catechol by bone marrow and lymphoid organs (1) can occur independently of the metabolism of benzene in these tissues and (2) is reduced under conditions in which the animal is less susceptible to benzene toxicity.

Correlation of regional formaldehyde flux predictions with the distribution of formaldehyde-induced squamous metaplasia in F344 rat nasal passages.

Squamous epithelium lines the nasal vestibule of the rat, rhesus monkey, and human. Respiratory, transitional, and olfactory epithelia line most areas posterior to the nasal vestibule. Inhaled formaldehyde gas induces squamous metaplasia posterior to the nasal vestibule and does not induce lesions in the nasal vestibule in rats and rhesus monkeys, indicating that squamous epithelium is resistant to irritant effects of formaldehyde and that squamous metaplasia may be an adaptive response. If squamous metaplasia is determined by formaldehyde dosimetry rather than by tissue-specific factors, squamous epithelium may be protective by absorbing less formaldehyde than other epithelial types. In a previous study, a three-dimensional, anatomically accurate computational fluid dynamics (CFD) model of the anterior F344 rat nasal passages was used to simulate inspiratory airflow and inhaled formaldehyde transport. The present study consisted of two related parts. First, the rat CFD model was used to test the hypothesis that the distribution of formaldehyde-induced squamous metaplasia is related to the location of high-flux regions posterior to squamous epithelium. Regional formaldehyde flux into nonsquamous epithelium predicted by the CFD model correlated with regional incidence of formaldehyde-induced squamous metaplasia on the airway perimeter of one cross-sectional level of the noses of F344 rats exposed to 10 and 15 ppm formaldehyde gas for 6 months. Formaldehyde flux into nonsquamous epithelium was estimated to vary by an order of magnitude depending on the degree of formaldehyde absorption by squamous epithelium. These results indicate that the degree to which squamous epithelium absorbs formaldehyde strongly affects the rate and extent of the progression of squamous metaplasia with continued exposure to formaldehyde. In the second part of this study, the CFD model was used to predict squamous metaplasia progression. Data needs for verification of this model prediction are considered. These results indicate that information on the permeability of squamous epithelium in rats, monkeys, and humans is important for accurate prediction of uptake in regions posterior to the nasal vestibule.

Application of Physiological Computational Fluid Dynamics Models to Predict Interspecies Nasal Dosimetry of Inhaled Acrolein

Acrolein is a highly soluble and reactive aldehyde and is a potent upper-respiratory-tract irritant. Acrolein-induced nasal lesions in rodents include olfactory epithelial atrophy and inflammation, epithelial hyperplasia, and squamous metaplasia of the respiratory epithelium. Nasal uptake of inhaled acrolein in rats is moderate to high, and depends on inspiratory flow rate, exposure duration, and concentration. In this study, anatomically accurate three-dimensional computational fluid dynamics (CFD) models were used to simulate steady-state inspiratory airflow and to quantitatively predict acrolein tissue dose in rat and human nasal passages. A multilayered epithelial structure was included in the CFD models to incorporate clearance of inhaled acrolein by diffusion, blood flow, and first-order and saturable metabolic pathways. Kinetic parameters for these pathways were initially estimated by fitting a pharmacokinetic model with a similar epithelial structure to time-averaged acrolein nasal extraction data and were then further adjusted using the CFD model. Predicted air:tissue flux from the rat nasal CFD model compared well with the distribution of acrolein-induced nasal lesions from a subchronic acrolein inhalation study. These correlations were used to estimate a tissue dose-based no-observed-adverse-effect level (NOAEL) for inhaled acrolein. A human nasal CFD model was used to extrapolate effects in laboratory animals to human exposure conditions on the basis of localized tissue dose and tissue responses. Assuming that equivalent tissue dose will induce similar effects across species, a NOAEL human equivalent concentration for inhaled acrolein was estimated to be 8 ppb.

Responses of the nasal mucociliary apparatus of F-344 rats to formaldehyde gas

The nasal mucociliary apparatus is an important component of the airway defenses. Studies were undertaken to determine the nature and distribution of acute effects of inhaled formaldehyde on the nasal mucociliary apparatus of male F-344 rats using whole body exposures. Formaldehyde exposures ranged from a single 6-hr period up to multiple 6-hr exposures daily for 3 weeks, with exposure concentrations of 15, 6, 2, 0.5, and 0 ppm. Within 1 hr of the last exposure, the rats were killed and the nasal passages examined for effects on nasal mucociliary function. Exposure to 15 ppm formaldehyde induced inhibition of mucociliary function in specific regions of the nose, and mucostasis was generally more extensive than ciliastasis. These effects, which were initially confined to the anterior regions of the nose, became progressively more extensive for up to 2 weeks of exposure with only very slight progression during the third week. Inhibition of mucociliary function was much less severe with 6 ppm, minimal at 2 ppm, and not detected in rats following exposure to 0.5 ppm. The distribution of epithelial lesions, identified by histopathology, correlated well with the distribution of defective mucociliary function, but mucociliary function was a more sensitive indicator of toxicity. Localized defects in mucociliary function represent a potentially important consequence of exposure to formaldehyde.

Carcinogenicity and Toxicity of Inhaled Nitrobenzene in B6C3F1 Mice and F344 and CD Rats

The potential carcinogenicity and toxicity of inhaled nitrobenzene were evaluated following chronic (2-year) exposure in mice and rats. Male and female B6C3F1 mice were exposed to 0, 5, 25, or 50 ppm nitrobenzene, while male and female F344 rats and male CD rats were exposed to 0, 1, 5, or 25 ppm nitrobenzene. All exposures were for 6 hr/day, 5 days/week excluding holidays, for a total of 505 days over 2 years. Survival was not adversely affected by nitrobenzene exposure, and only mild exposure-related decreases in body weights (< 10% of control) were occasionally noted. Nitrobenzene exposure resulted in increased incidence of neoplasia in male B6C3F1 mice (pulmonary alveolar/bronchiolar and thyroid follicular cell neoplasms), female B6C3F1 mice (mammary gland neoplasms), male F344 rats (hepatocellular and renal neoplasms), female F344 rats (endometrial stromal neoplasms), and male CD rats (hepatocellular neoplasms). In addition, there were marginal increases in the incidence of hepatocellular neoplasia in female B6C3F1 mice and thyroid follicular neoplasia in male F344 rats. Groups of nitrobenzene-exposed mice and rats with increased incidence of renal and thyroid neoplasia also had increased incidences of hyperplasia in these tissues. Toxicity resulting from chronic inhalation of nitrobenzene was manifested by methemoglobinemia, anemia, and adaptive or degenerative changes in the nose, liver, and testis. The results indicate that inhaled nitrobenzene is carcinogenic and toxic in mice and rats, and that the spectrum of these responses in animals is dependent on species, sex, and genetic background.

Distribution, progression, and recovery of acute formaldehyde-induced inhibition of nasal mucociliary function in F-344 rats☆

A previous report of inhalation exposure of F-344 rats to formaldehyde gas, using a whole-body exposure system, described the induction of regional inhibition of nasal mucociliary function, with a clear concentration-response relationship. A head-only exposure system was subsequently developed in order to facilitate the present study of reversibility of acute effects of formaldehyde on the nasal mucociliary apparatus. This study also included an examination of more extensive areas of the nose than those reported in the previous work. Male F-344 rats were exposed to 2 or 15 ppm formaldehyde gas for 10, 20, 45, or 90 min or 6 hr with recovery groups examined 1 hr after the end of the 90-min and 6-hr exposures. No effects were observed in rats exposed to 2 ppm formaldehyde. In rats exposed to 15 ppm, the extent of formaldehyde-induced inhibition of mucociliary function detected in specific regions of the nose was time dependent, with increasing areas of mucostasis and ciliastasis being induced during a 6-hr exposure period. A 1-hr room-air exposure, following exposure to 15 ppm formaldehyde, resulted in marked recovery of mucociliary function, indicating the value of a head-only exposure system for rapid examination of mucociliary function following exposure. Recovery of mucociliary function occurred especially in the more posterior areas of affected regions of the nose. However, in areas of recovery mucus flow rate was reduced compared to unexposed control rates, indicating incomplete recovery of function in these areas. Regions of formaldehyde-induced inhibition of mucociliary function correlated well with the previously reported distribution of formaldehyde-induced nasal squamous cell carcinomas, with the exception of effects on the medial aspect of the maxilloturbinate. These findings were considered to provide further support for the proposal that both regional exposure and local tissue susceptibility may be responsible for the distribution of formaldehyde-induced nasal squamous cell carcinomas. It was also postulated, on the basis of mucus flow patterns derived from control animals in this study, that flow relationships between nasal mucus and inspired air form a countercurrent system which may optimize clearance of inhaled air contaminants.

Two-Year Inhalation Exposure of Female and Male B6C3F1 Mice and F344 Rats to Chlorine Gas Induces Lesions Confined to the Nose

Chlorine gas is a respiratory irritant in both animals and humans that produces concentration-dependent responses ranging from minor irritation to death. Female and male B6C3F1 mice and F344 rats were exposed to chlorine gas for up to 2 years to determine chronic toxicity and carcinogenicity. Groups of approximately 70 each of female and male mice and rats were exposed to 0, 0.4, 1.0, or 2.5 ppm chlorine gas for 6 hr/day, 5 days/week (mice and male rats), or 3 alternate days/week (female rats) for 2 years, with an interim necropsy of rats at 12 months (10 rats/sex/concentration group). A complete necropsy was performed on all animals. Histological examination was performed on all organs from high-concentration and control animals and selected target organs from mid- and low-concentration groups. Exposure-dependent lesions were confined to the nasal passages in all sex and species groups. Chlorine-induced lesions, which were most severe in the anterior nasal cavity, included respiratory and olfactory epithelial degeneration, septal fenestration, mucosal inflammation, respiratory epithelial hyperplasia, squamous metaplasia and goblet cell hypertrophy and hyperplasia, and secretory metaplasia of the transitional epithelium of the lateral meatus. Intracellular accumulation of eosinophilic proteinaceous material was also a prominent response involving the respiratory, transitional, and olfactory epithelia, and in some cases the squamous epithelium of the nasal vestibule. Many of these nasal lesions exhibited an increase in incidence and/or severity that was related to chlorine exposure concentration and were statistically significantly increased at all chlorine concentrations studied. Male mice and female rats appeared more sensitive to chlorine than female mice and male rats, respectively. The reasons for the sex differences within a species were not determined. Interspecies differences in regional dosimetry and site-specific tissue susceptibility to chlorine exposure should be taken into account when using these data for accurate assessment of potential human health risks. The incidence of neoplasia was not increased by exposure, indicating that inhaled chlorine in rats and mice is an upper respiratory tract toxicant but not a carcinogen.