Respiratory Tract Toxicity Research Articles

Use of bronchoalveolar lavage to detect respiratory tract toxicity of inhaled material

The first epithelial surface encountered by inhaled materials is the epithelium of the respiratory tract. The epithelium is lined by a fluid (ELF) that can be sampled by a saline wash (lavage) of the area of interest. This technique, known as bronchoalveolar lavage (BAL), provides a means of sampling a body fluid that can provide valuable information on the reaction of the lung to inhaled materials. The most common responses measured are indicators of an inflammatory response, the most sensitive of which is an influx of neutrophils. In the extracellular fluid, levels of beta-glucuronidase activity indicate activation of macrophages, and lactate dehydrogenase activity indicates cytotoxicity. Other pro- and anti-inflammatory soluble factors that can be measured in BAL fluid include secretory products of macrophages and epithelial cells, such as tumor necrosis factor alpha, fibronectin, interleukin-1, various chemotactic factors (including IL-8, MIP-2), growth factors, proteases, and antiproteases. Oxidative stress can be measured by the levels of reduced glutathione in ELF, and increased levels of alkaline phosphatase indicate increased Type II cell secretions. Allergic responses are indicated by increased eosinophils and factors such as histamine and arachidonate metabolites in BAL fluid. BAL analysis can be used as a complementary technique with more traditional measures of lung injury, such as histopathology or radiology. The advantage of BAL analysis is that one can pick up early indicators of biochemical changes leading to later morphological changes in a disease process. A second advantage is that the BAL fluid analyses are quantitative, and dose–response measures can be obtained. In large animals, one can do repeated lavages to follow a disease process; in small animals, one can use serial sacrifices in similarly exposed rodents to achieve the same goal. Research related to the use of BAL fluid analyses to detect lung damage has been conducted at the Lovelace Respiratory Research Institute with funding from various sources including the US Department of Energy and the US Environmental Protection Agency.

Induction of heat shock protein 70 in rat olfactory epithelium by toxic chemicals: in vitro and in vivo studies

We have previously developed a rat nasal explant system for investigating upper respiratory tract toxicity, and the aims of this study were to determine whether heat shock protein (HSP) 70 is induced in this model following exposure to carbon tetrachloride (CCl4), dimethyl adipate (DMA), methyl iodide (CH3I) or paracetamol, and whether HSP70 can also be induced in the nasal cavity in vivo. Intracellular ATP was significantly depleted in ethmoturbinates incubated for 4 h with the toxins (0-100 mM; EC50 concentrations: CCl4 32 mM, DMA 3 mM, CH3I 1.5 mM, paracetamol 70 mM), but there was little induction of HSP70. Turbinates were then incubated for 1 h with CCl4 (5 mM), DMA (1.5 mM), CH3I (0.57 mM) or paracetamol (30 mM) and allowed to recover for up to 24 h. Treatment with CCl4, DMA or paracetamol resulted in 250-300% induction of HSP70. Male rats were administered a single oral dose of CCl4 (1600 mg/kg) and killed 16 h later. Degenerative lesions (epithelial undulation and hydropic vacuolation) were evident in the olfactory epithelium, and immunohistochemical analysis of HSP70 revealed increased staining in, or proximate to, areas of damage. Thus, HSP70 can be induced in the olfactory epithelium both in vitro and in vivo.

BM-derived cells restore expression of peroxiredoxin V in the airways following acute naphthalene injury in mice

Naphthalene-induced respiratory tract toxicity in mice is characterized by specific and rapid loss of the Clara cell population, which is restored only after several days. The sources of restoration of this cell population remain unclear. We investigated whether BM-derived cells participated in the process of epithelial restoration following naphthalene toxicity compared with bacterial infection. We further investigated the role of BM-derived cells in restoration of expression of peroxiredoxin V (PRXV), one of the major proteins of antioxidant defense, specifically expressed in the bronchial epithelium. We transplanted GFP-tagged BM cells into 5 Gy-irradiated C57BL/6 recipients. Following 1 month of recovery, experimental animals were subjected to 250 mg/kg naphthalene i.p. An additional group of animals received intratracheal instillation of Escherichia coli to induce acute bacterial inflammation. Animals were killed at 1-12 days after naphthalene and analyzed immunohistochemically. Recipients' cells of bronchial epithelium demonstrated significantly reduced levels of PRXV expression following naphthalene. In animals with acute bacterial inflammation, PRXV levels were not reduced in epithelium and participation of BM-derived cells in epithelial restoration was minimal. Following naphthalene, GFP(+) cells were present in large numbers in lung parenchyma and epithelium of conducting airways starting at 1 day following injury. GFP(+) progeny of BM cells was the major source of PRXV in the epithelium. These data suggest that BM-derived cells may provide a source of antioxidant protection of airways by expression of PRXV in a model of acute epithelial respiratory tract toxicity.

Respiratory tract toxicity of inhaled hydrogen sulfide in Fischer-344 rats, Sprague–Dawley rats, and B 6C 3F 1 mice following subchronic (90-day) exposure

The goal of this study was to characterize the toxicity of hydrogen sulfide (H 2S), including nasal and pulmonary effects, in adult male and female Fischer-344 and Sprague–Dawley rats and B 6C 3F 1 mice. Animals underwent whole-body exposure to 0, 10, 30, or 80 ppm H 2S for 6 h/day for at least 90 days. Exposure to 80 ppm H 2S was associated with reduced feed consumption during either the first exposure week (rats) or throughout the 90-day exposure (mice). Male Fischer-344 rats, female Sprague–Dawley rats, and female B 6C 3F 1 mice exposed to 80 ppm H 2S had depressed terminal body weights when compared with air-exposed controls. Subchronic H 2S inhalation did not result in toxicologically relevant alterations in hematological indices, serum chemistries, or gross pathology. Histologic evaluation of the nose showed an exposure-related increased incidence of olfactory neuronal loss (ONL) and rhinitis. ONL occurred following exposure to ≥30 ppm H 2S in both sexes of all experimental groups, with one exception, male Sprague–Dawley rats demonstrated ONL following exposure to 80 ppm H 2S only. A 100% incidence of rhinitis was found in the male and female B6C3F1 mice exposed to 80 ppm H 2S. In the lung, exposure to H 2S was associated with bronchiolar epithelial hypertrophy and hyperplasia in male and female Sprague–Dawley rats following exposure to ≥30 ppm H 2S and in male Fischer-344 rats exposed to 80 ppm H 2S. Our results confirm that the rodent nose, and less so the lung, are highly sensitive to H 2S-induced toxicity, with 10 ppm representing the NOAEL for ONL following subchronic inhalation.

PbCrO4 mediates cellular responses via reactive oxygen species.

Exposure to certain particulate hexavalent chromium [Cr(VI)] compounds, such as lead chromate (PbCrO4), has been associated with lung cancer and respiratory tract toxicity. Previous studies indicate that the solubility of Cr(VI)-compounds is an important factor in Cr(VI)-induced carcinogenesis. The present study investigates reactive oxygen species (ROS) generation by PbCrO4 particles and cellular responses using RAW 264.7 cells. A mixture containing PbCrO4 and RAW 264.7 cells generated hydroxyl radical ((.)OH), using cellularly generated H2O2 as a precursor, as measured by electron spin resonance (ESR) spin trapping in combination with H2O2 and (.)OH scavengers, catalase and sodium formate. The effect of ascorbic acid on (.)OH radicals was also measured using ESR. Confocal microscopy showed that particles could become either bound to the cell surface or engulfed over a 120 min time period. H2O2 generation and O2 consumption were also increased after treatment of the cells with PbCrO4. Both NF-kappaB and AP-1 were activated after exposure to PbCrO4 particles as measured by the NF-kappaB or AP-1 luciferase reporter plasmid assay. Our investigation thus demonstrated that the RAW 264.7 cells phagocytized the PbCrO4 particles leading to accumulation of the particles within vacuoles in the cytoplasm. These particles could induce chronic production of ROS and activation of NF-kappaB and AP-1. Such induction of transcription pathways may be involved in the inflammatory and carcinogenic responses induced by Cr(VI)-containing particles.

Physiologically-based kinetic modeling of vapours toxic to the respiratory tract

The respiratory tract is frequently identified as a site of toxicity for inhaled xenobiotic chemicals. Usually, these observations come from controlled animal studies. For these studies to be of quantitative value to human health risk assessment, species-specific factors governing dosimetry of inhaled substances must be taken into account. Toxicokinetics of vapours in the respiratory tract are defined by absorption, distribution, metabolism, and excretion, as they are in other tissues; however, these concepts take on new dimensions when considering respiratory tract toxicants, especially those that elicit portal of entry effects by directly interacting with the tissue lining the respiratory tract. Species-specific factors related to anatomy, physiology and biochemistry govern inter-species extrapolation of toxicokinetics. This article discusses critical factors of respiratory tract kinetics that should be considered when developing physiological-based toxicokinetic (PBTK) models for inhaled vapours. Important considerations such as impact of regional airflow-delivery, water solubility, reactivity, and rates of local biotransformation on respiratory tract tissue dosimetry are highlighted. These factors can be accounted for only to a limited extent when using default approaches to extrapolate dosimetry of inhaled substances across species. On the other hand, PBTK modeling has the flexibility to accommodate many of the critical determinants of respiratory tract toxicity. PBTK models can also help identify the most critical toxicokinetic data necessary to replace defaults. PBTK approaches have led to more informed estimates of human target tissue dose, and therefore human health risk, especially where these risk assessments have been based on extrapolation of animal dosimetry studies. Experience derived from the development of more intensive case studies have, in turn, enabled simplified approaches to the use of PBTK modeling for respiratory tract toxicants. Whether simplified or highly complex, PBTK modeling approaches are proven to be of great utility to risk assesors interested in applying quantitative information to informed risk assessment evaluations.

Identification of glutathione modifications by cigarette smoke.

Although it has been recognized for decades that cigarette smoke (CS) is toxic to respiratory tract tissues, and that glutathione (GSH) and other thiols are able to ameliorate some of the adverse effects of CS, the precise interactions between thiols and critical CS components are only partially characterized. In the present study, we used HPLC and MALDI-MS approaches to more rigorously characterize the products of CS reactions with GSH, the major cellular thiol and an important antioxidant constituent in respiratory tract lining fluids, in an attempt to increase our understanding of mechanisms of CS respiratory tract toxicity. Exposure of solutions of GSH to gas phase CS resulted in its rapid depletion, and about 50% of this depletion could be accounted for by reaction with acrolein and crotonaldehyde, the two major alpha, beta-unsaturated aldehydes in CS. Similar aldehyde adducts with GSH could also be detected in cells exposed to CS, although the relative yields were limited, presumably because of further reactions of these adducts and/or their excretion. Further characterization of in vivo thiol-aldehyde formation in respiratory tract cells can be expected to provide significant insights into the mechanisms of CS toxicity.

Styrene Respiratory Tract Toxicity and Mouse Lung Tumors Are Mediated by CYP2F-Generated Metabolites

Mice are particularly sensitive to respiratory tract toxicity following styrene exposure. Inhalation of styrene by mice results in cytotoxicity in terminal bronchioles, followed by increased incidence of bronchioloalveolar tumors, as well as degeneration and atrophy of nasal olfactory epithelium. In rats, no effects on terminal bronchioles are seen, but effects in the nasal olfactory epithelium do occur, although to a lesser degree and from higher exposure concentrations. In addition, cytotoxicity and tumor formation are not related to blood levels of styrene or styrene oxide (SO) as measured in chronic studies. Whole-body metabolism studies have indicated major differences in styrene metabolism between rats and mice. The major differences are 4- to 10-fold more ring-oxidation and phenylacetaldehyde pathways in mice compared to rats. The data indicate that local metabolism of styrene is responsible for cytotoxicity in the respiratory tract. Cytotoxicity is seen in tissues that are high in CYP2F P450 isoforms. These tissues have been demonstrated to produce a high ratio of R-SO compared to S-SO (at least 2.4 : 1). In other rat tissues the ratio is less than 1, while in mouse liver the ratio is about 1.1. Inhibition of CYP2F with 5-phenyl-1-pentyne prevents the styrene-induced cytotoxicity in mouse terminal bronchioles and nasal olfactory epithelium. R-SO has been shown to be more toxic to mouse terminal bronchioles than S-SO. In addition, 4-vinylphenol (ring oxidation of styrene) has been shown to be highly toxic to mouse terminal bronchioles and is also metabolized by CYP2F. In human nasal and lung tissues, styrene metabolism to SO is below the limit of detection in nearly all samples, and the most active sample of lung was approximately 100-fold less active than mouse lung tissue. We conclude that styrene respiratory tract toxicity in mice and rats, including mouse lung tumors, are mediated by CYP2F-generated metabolites. The PBPK model predicts that humans do not generate sufficient levels of these metabolites in the terminal bronchioles to reach a toxic level. Therefore, the postulated mode of action for these effects indicates that respiratory tract effects in rodents are not relevant for human risk assessment.

Upper respiratory tract toxicity of inhaled methylvinyl ketone in F344 rats and B6C3F1 mice.

The National Toxicology Program is conducting a chemical class study to investigate the structure-activity relationships for the toxicity of alpha,beta-unsaturated ketones. Methylvinyl ketone (MVK) was selected for study because it is a representative straight-chain aliphatic alpha,beta-unsaturated ketone and because of its extensive use and widespread exposure. Short-term inhalation studies of MVK were conducted to provide toxicity data for comparison with other alpha,beta-unsaturated ketones and for use in designing chronic toxicity and carcinogenicity studies. In 2-week studies, rats and mice were exposed to 0, 0.25, 0.5, 1, 2, 4, or 8 ppm MVK 6 h/day, 5 days/week for 12 exposures. Morbidity and early deaths occurred in all male and female rats after 1 exposure and in 2 male mice after 10 exposures to 8 ppm. Rats exhibited nasal cavity toxicity and lung necrosis at 4 ppm. No toxicity was observed in animals exposed to less than 2 ppm. Based on these results a 13-week study was conducted at 0, 0.5, 1, and 2 ppm MVK. As observed in the 2-week study, the nasal cavity was the main target organ and rats were more sensitive than mice. Respiratory and olfactory epithelial necrosis were prominent by day 21 in the rat. At study termination these lesions were still evident but not as severe as noted earlier. Additionally, changes such as olfactory epithelial regeneration and metaplasia (respiratory) as well as respiratory epithelial hyperplasia and metaplasia (squamous) were clearly evident. Nasal lesions in mice were limited to a subtle squamous metaplasia of transitional and/or respiratory epithelium covering predominantly the tips of naso- and maxilloturbinates in Levels I and II. A transient, leukopenia was observed in rats exposed to 2 ppm, however, this effect was not present after 13 weeks of exposure. In mice, leukocyte counts were significantly decreased at all exposure concentrations after 13 weeks of exposure. Absolute testicular and epididymal weights and sperm counts were decreased at the high dose only. MVK can be characterized as a reactive, direct-acting gaseous irritant. MVK exposure causes the same nasal cavity lesions as the cyclic alpha,beta-unsaturated ketone, 2-cyclohexen-1-one, although at lower exposure concentrations.

Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations.

The evaluation of respiratory tract toxicity from airborne materials frequently involves exposure of animals via inhalation. This provides a natural route of entry into the host and, as such, is the preferred method for the introduction of toxicants into the lungs. However, for various reasons, this technique cannot always be used, and the direct instillation of a test material into the lungs via the trachea has been employed in many studies as an alternative exposure procedure. Intratracheal instillation has become sufficiently widely used that the Inhalation Specialty Section of the Society of Toxicology elected to develop this document to summarize some key issues concerning the use of this exposure procedure. Although there are distinct differences in the distribution, clearance, and retention of materials when administered by instillation compared to inhalation, the former can be a useful and cost-effective procedure for addressing specific questions regarding the respiratory toxicity of chemicals, as long as certain caveats are clearly understood and certain guidelines are carefully followed.

A rat nasal epithelial model for predicting upper respiratory tract toxicity: in vivo–in vitro correlations

An in vitro model of the rat nasal cavity has been used to compare the responses of nasal tissues in vitro, using loss of intracellular ATP and potassium as indices of toxicity, with the pathological changes occurring following in vivo exposure to four test compounds. Turbinates were incubated in vitro with the test compounds for 4 h, for 24 h or for 4 h followed by 20 h in fresh medium. Titanium dioxide caused little or no loss of ATP in either olfactory epithelium (OE) or respiratory epithelium (RE). Sodium carbonate decreased olfactory, but not respiratory ATP, while acetic acid and 3-methylindole markedly decreased ATP in both tissues. Intracellular potassium concentrations were generally affected to a lesser degree. In vivo, no morphological changes were observed in the nasal cavity following inhalation exposure to either titanium dioxide or sodium carbonate. Inhalation of acetic acid resulted in a very focal lesion in the RE of the dorsal meatus of level 1, while administration of 3-methylindole by intraperitoneal injection caused severe degeneration of OE. In further experiments olfactory turbinates were exposed to a range of concentrations (0–100 mM) of sodium carbonate, acetic acid and 3-methylindole for 4 h and ATP concentrations determined. Concentration-dependent decreases in ATP were observed for sodium carbonate and 3-methylindole, with EC 50 values estimated as 2.57 and 0.91 mM, respectively. Acetic acid only decreased ATP significantly at the 100-mM concentration. In summary, this in vitro model has predicted the nasal toxicity of several compounds, including both direct-acting agents (sodium carbonate, acetic acid) and one requiring metabolic activation (3-methylindole). However, the lack of airflow-dependent dosimetry, results in some lack of discrimination between the different regions of the nasal cavity and may make this model overly sensitive.

Internalization of Carcinogenic Lead Chromate Particles by Cultured Normal Human Lung Epithelial Cells: Formation of Intracellular Lead-Inclusion Bodies and Induction of Apoptosis

Occupational exposure to certain particulate hexavalent chromium [Cr(VI)] compounds, such as lead chromate, has been associated with lung cancer and respiratory tract toxicity. We have previously shown that apoptosis is a major mode of death in cultured rodent cells treated with soluble sodium chromate and particulate lead chromate. Here we report the cellular and molecular effects of lead chromate and sodium chromate in normal human lung small airway epithelial (HSAE) cells, which may be one of the targets for Cr(VI)-induced lung cancer and respiratory tract toxicity. Phagocytosed lead chromate particles and intracellular lead-inclusion bodies (LIB) were observed by transmission electron microscopy and confirmed by X-ray analysis. HSAE cells exposed to lead chromate and sodium chromate underwent dose-dependent apoptosis. The cellular uptake and genomic interactions of both Cr and lead (Pb) were examined by inductively coupled plasma mass spectrometry (ICPMS) coupled with a novel, direct-injection high-efficiency nebulizer (DIHEN). Using this approach, we have quantitated a dose-dependent formation of Cr-DNA adducts and DNA-associated Pb in lead chromate–treated HSAE cells. The formation of LIB in normal human lung cells exposed to lead chromate indicates that ionic Pb is released from the particles and thus might contribute to the cell toxicity caused by lead chromate. Internalization and dissolution of lead chromate particles and the interaction of ionic Cr and Pb with DNA, may be components of the mechanism of lead chromate carcinogenesis. Lead chromate–induced apoptosis may be a mechanism to eliminate cells with chromium- and/or lead-damaged DNA.

Peripheral markers (Clara cell protein and alpha-glutathione S-transferase) and lipidoperoxidation (malondialdehyde) assessment in Sprague-Dawley rats instilled with haematite and benzo[a]pyrene.

The literature suggests that the concomitant exposure to polycyclic aromatic hydrocarbons (PAH) and ferric oxide particles could enhance lung cancer incidence in environmental and occupational settings. High levels of tracheobronchial tumours were obtained in hamsters exposed to benzo[a]pyrene (B[a]P) adsorbed onto ferric oxide carrier particles. Therefore, we have assessed the toxic effects of exposure to haematite (Fe2O3) and B[a]P in male Sprague-Dawley rats. Animals were instilled with the chemicals alone (3 mg of Fe2O3 or B[a]P) or in combination (3 mg Fe2O3 + 3 mg B[a]P). Bronchoalveolar lavages (BAL) and biological samples (serum and urine) were collected 48 h after the intoxication. Clara cell protein (CC16) and alpha-glutathione S-transferase (alpha-GST), as peripheral markers of both tracheobronchial epithelial cell integrity and renal dysfunction, were determined in BAL fluid, serum and urine. Malondialdehyde (MDA), a marker of lipid peroxidation, was measured in BAL fluid and serum. We observed a significant increase of CC16 concentrations in BAL fluid after Fe2O3 + B[a]P instillation (p < 0.05) in serum after Fe2O3 and Fe2O3 + B[a]P exposure (p < 0.01) and in urine after B[a]P administration (p < 0.01). Instillation of Fe2O3 + B[a]P produced an increased amount of alpha-GST in BAL fluid (p < 0.01), whereas B[a]P alone caused a significant elevation of alpha-GST in serum and urine (p < 0.01). Moreover, Fe2O3 or Fe2O3 + B[a]P instillation induced a significant increase in MDA levels in BAL fluid (p < 0.01 and p < 0.05). In conclusion, Fe2O3 may have a low pulmonary toxicity. However, B[a]P manifested a rapid and high toxicity in the respiratory tract and kidneys. When B[a]P was adsorbed on haematite particles, both its retention in the respiratory tract and pulmonary toxicity increased.

Evaluation of the inhalation toxicity of diethylethanolamine (DEEA) in rats

Diethylethanolamine (DEEA), an industrial anticorrosive additive, was evaluated in rats for its potential toxicity. A 2-week inhalation exposure to 10 ppm revealed no signs of toxicity. At 56 ppm, rats exhibited signs of nasal irritation and corneal opacities. Significant mortality (males, 90%; females, 50%) occurred at 301 ppm. Histopathology revealed inflammatory cell infiltrations of the nasal turbinate mucosa and squamous metaplasia after exposure to 56 ppm. In the 14-week subchronic study, rats were exposed to 0, 11, 25, or 76 ppm. During exposure to DEEA, transient signs of mild to moderate respiratory irritation (noises or rales) were observed with a frequency and severity that was dose dependent. These signs usually cleared within 1 hr postexposure. However, at 76 ppm some rats continued to exhibit these respiratory signs overnight. There were no significant DEEA-induced changes in either blood chemistry or neurobehavioral parameters. Nasal cavities of rats exposed to 25 or 76 ppm revealed evidence of inflammatory cell infiltration, focal hyperplasia, and squamous metaplasia in the respiratory epithelium of the anterior nasal turbinates. Hypertrophic goblet cells, focal necrosis, and exudate in the nasal lumen were also seen at 76 ppm. DEEA vapors failed to produce persistent ocular or respiratory tract toxicity below 26 ppm. The no-observed-effect-level in this study was 10 ppm.

In vitro models of lung toxicity

In vitro assays that emphasize cellular components critical to the host defense system have been developed to evaluate pulmonary toxicity and define deleterious changes in parenchymal cell populations. Assays that employ pulmonary alveolar macrophages (PAM) have demonstrated good correlation between macrophage toxicity and pulmonary fibrogenicity for many inorganic compounds. The PAM assays provide simple and inexpensive screens of potential respiratory tract toxicity. Many investigators screen chemicals for their ability to alter the mucosal epithelium of the conducting airways by performing tracheal organ cultures. The tracheal assays have also provided useful screens for Vitamin A analogues required for epithelial cell differentiation. Most recently, in vitro respiratory tract models have been extended to include whole-lung explants, an approach that allows for development of fibrosis and epithelial cell toxicity after in vitro exposure to inorganic and organic fibrogens. The whole-lung explant system appears to duplicate the in vivo response to a variety of lung toxins, including bleomycin, silica, and crocidolite asbestos. Together, these assays provide a description of potential toxicity to key components of the lung, emphasizing the pulmonary macrophage, conducting airways, and alveolar septae. It is expected that continued research in these models will enhance their predictive abilities and utility in risk assessment.

Effect of single or repeated formaldehyde exposure on minute volume of B6C3F1 mice and F-344 rats

To characterize species differences in sensory irritation to formaldehyde (HCHO) and to determine if tidal volume ( V T) compensation parallels the respiratory rate (ƒ) depression, ƒ, V T, and minute (V̇ E) of B6C3F1 mice and F-344 rats were measured during 10-min, head-only HCHO exposures. Comparisons also were made between naive (no previous HCHO exposure) and pretreated animals (HCHO, 2, 6, or 15 ppm, 6 hr/day for 4 days). HCHO exposure elicited immediate and simultaneous decreases in ƒ and V̇ E in a concentration-dependent manner. No significant compensation in V T was observed in either species except the 15-ppm-pretreated rats. While ƒ and V̇ E remained maximally depressed in mice for the remainder of the exposure period, recovery occurred in rats. In addition to tolerance development, rats were also less sensitive to HCHO. The RD50 (concentration at which ƒ decreased by 50%) of naive rats was 31.7 ppm vs 4.9 ppm for mice. Similar RD50 values and time-response curves for V̇ E and ƒ were observed among naive and pretreated groups, although the baseline ƒ of pretreated animals was significantly depressed as compared to naive animals. In summary, HCHO exposure depressed V̇ E in both mice and rats. But, because of greater sensitivity and lack of tolerance, mice were able to minimize inhalation of HCHO more than rats. This species difference may contribute to differences in respiratory tract toxicity from inhaled HCHO.