A review of pulmonary neutrophilia and insights into the key role of neutrophils in particle-induced pathogenesis in the lung from animal studies of lunar dusts and other poorly soluble dust particles.

The mechanisms of particle-induced pathogenesis in the lung remain poorly understood. Neutrophilic inflammation and oxidative stress in the lung are hallmarks of toxicity. Some investigators have postulated that oxidative stress from particle surface reactive oxygen species (psROS) on the dust produces the toxicopathology in the lungs of dust-exposed animals. This postulate was tested concurrently with the studies to elucidate the toxicity of lunar dust (LD), which is believed to contain psROS due to high-speed micrometeoroid bombardment that fractured and pulverized lunar surface regolith. Results from studies of rats intratracheally instilled (ITI) with three LDs (prepared from an Apollo-14 lunar regolith), which differed 14-fold in levels of psROS, and two toxicity reference dusts (TiO2 and quartz) indicated that psROS had no significant contribution to the dusts' toxicity in the lung. Reported here are results of further investigations by the LD toxicity study team on the toxicological role of oxidants in alveolar neutrophils that were harvested from rats in the 5-dust ITI study and from rats that were exposed to airborne LD for 4 weeks. The oxidants per neutrophils and all neutrophils increased with dose, exposure time and dust's cytotoxicity. The results suggest that alveolar neutrophils play a critical role in particle-induced injury and toxicity in the lung of dust-exposed animals. Based on these results, we propose an adverse outcome pathway (AOP) for particle-associated lung disease that centers on the crucial role of alveolar neutrophil-derived oxidant species. A critical review of the toxicology literature on particle exposure and lung disease further supports a neutrophil-centric mechanism in the pathogenesis of lung disease and may explain previously reported animal species differences in responses to poorly soluble particles. Key findings from the toxicology literature indicate that (1) after exposures to the same dust at the same amount, rats have more alveolar neutrophils than hamsters; hamsters clear more particles from their lungs, consequently contributing to fewer neutrophils and less severe lung lesions; (2) rats exposed to nano-sized TiO2 have more neutrophils and more severe lesions in their lungs than rats exposed to the same mass-concentration of micron-sized TiO2; nano-sized dust has a greater number of particles and a larger total particle-cell contact surface area than the same mass of micron-sized dust, which triggers more alveolar epithelial cells (AECs) to synthesize and release more cytokines that recruit a greater number of neutrophils leading to more severe lesions. Thus, we postulate that, during chronic dust exposure, particle-inflicted AECs persistently release cytokines, which recruit neutrophils and activate them to produce oxidants resulting in a prolonged continuous source of endogenous oxidative stress that leads to lung toxicity. This neutrophil-driven lung pathogenesis explains why dust exposure induces more severe lesions in rats than hamsters; why, on a mass-dose basis, nano-sized dusts are more toxic than the micron-sized dusts; why lung lesions progress with time; and why dose-response curves of particle toxicity exhibit a hockey stick like shape with a threshold. The neutrophil centric AOP for particle-induced lung disease has implications for risk assessment of human exposures to dust particles and environmental particulate matter.

Comparative pulmonary toxicities of lunar dusts and terrestrial dusts (TiO2 & SiO2) in rats and an assessment of the impact of particle-generated oxidants on the dusts' toxicities.

Humans will set foot on the Moon again soon. The lunar dust (LD) is potentially reactive and could pose an inhalation hazard to lunar explorers. We elucidated LD toxicity and investigated the toxicological impact of particle surface reactivity (SR) using three LDs, quartz, and TiO2. We first isolated the respirable-size-fraction of an Apollo-14 regolith and ground two coarser samples to produce fine LDs with increased SR. SR measurements of these five respirable-sized dusts, determined by their in-vitro ability to generate hydroxyl radicals (•OH), showed that ground LDs > unground LD ≥ TiO2 ≥ quartz. Rats were each intratracheally instilled with 0, 1, 2.5, or 7.5 mg of a test dust. Toxicity biomarkers and histopathology were assessed up to 13 weeks after the bolus instillation. All dusts caused dose-dependent-increases in pulmonary lesions and toxicity biomarkers. The three LDs, which possessed mineral compositions/properties similar to Arizona volcanic ash, were moderately toxic. Despite a 14-fold •OH difference among these three LDs, their toxicities were indistinguishable. Quartz produced the lowest •OH amount but showed the greatest toxicity. Our results showed no correlation between the toxicity of mineral dusts and their ability to generate free radicals. We also showed that the amounts of oxidants per neutrophil increased with doses, time and the cytotoxicity of the dusts in the lung, which supports our postulation that dust-elicited neutrophilia is the major persistent source of oxidative stress. These results and the discussion of the crucial roles of the short-lived, continuously replenished neutrophils in dust-induced pathogenesis are presented.

Characterization of biochemical, functional and structural changes in mice respiratory organs chronically exposed to cigarette smoke.

Female C57BL/6 mice were exposed to mainstream cigarette smoke at 600 µg WTPM/L, 4 h/day and 5 days/week for up to 52 weeks. At 26, 52 and 65 weeks (52 weeks of exposure plus 13 weeks of no exposure), lungs were assessed for inflammation, function, histopathology and morphometry. Structural changes were observed and accompanied by altered lung function at 26 and 52 weeks (e.g. increase of static compliance and hysteresis, and decrease of elastance). Lung morphometry quantified significant increase in airspace enlargement at 52 weeks. Chronic smoke exposure induced inflammation in respiratory organs, e.g. mixed inflammatory cell infiltrates, perivascular lymphocyte infiltrates and pigmented alveolar macrophages in the lungs. Minimal or mild alveolar emphysema was diagnosed in 70% by 26 weeks or 80% by 52 weeks. After 13 weeks of recovery, most biochemical, histopathological and morphometrical alterations were restored, while emphysema was observed to persist at 18% incidence by 65 weeks. In conclusion, the employed exposure conditions induced emphysematous changes in the lungs, accompanied by altered lung function and morphological/histopathological changes. Following the 13 weeks of no exposure, morphological changes persisted, although some functional/biochemical alterations regressed.

Biological responses in rats exposed to mainstream smoke from a heated cigarette compared to a conventional reference cigarette.

The heated cigarette (HC) generates mainstream smoke by vaporizing the components of the tobacco rod using a carbon heat source at the cigarette tip. Mainstream smoke of HC contains markedly less chemical constituents compared to combusted cigarettes. Mainstream smoke from HC was generated under Health Canada Intense regimen and its biological effects were compared to those of Reference (3R4F) cigarettes, using nose-only 5-week and 13-week inhalation studies. In the 13-week study, SD rats were necropsied following exposure to mainstream smoke from each cigarette at 200, 600 or 1000 µg wet total particulate matter/L for 1 h/day, 7 days/week or following a 13-week recovery period. Histopathological changes in the respiratory tract were significantly lesser in HC groups; e.g. respiratory epithelial hyperplasia in the nasal cavity and accumulation of pigmented macrophages in alveoli. After a 13-week recovery, the lesions were completely or partially regressed, except for accumulation of pigmented macrophages in alveoli, in both HC and 3R4F groups. In the 5-week study, SD rats were necropsied following exposure to mainstream smoke of either cigarette at 600 or 1000 µg/L for 1 h, two times/day (with 30 min interval), 7 days/week or following a 4-week recovery period. Bronchoalveolar lavage fluid (BALF) analysis of neutrophil percentages and enzyme levels like γ-GT, ALP and LDH indicated that pulmonary inflammation was significantly less in HC groups compared to 3R4F groups. In conclusion, HC demonstrated significantly lower biological effects compared to 3R4F, based on the BALF parameters and histopathology.

Toxicity of lunar dust assessed in inhalation-exposed rats.

Humans will again set foot on the moon. The moon is covered by a layer of fine dust, which can pose a respiratory hazard. We investigated the pulmonary toxicity of lunar dust in rats exposed to 0, 2.1, 6.8, 20.8 and 60.6 mg/m(3) of respirable-size lunar dust for 4 weeks (6 h/day, 5 days/week); the aerosols in the nose-only exposure chambers were generated from a jet-mill ground preparation of a lunar soil collected during the Apollo 14 mission. After 4 weeks of exposure to air or lunar dust, groups of five rats were euthanized 1 day, 1 week, 4 weeks or 13 weeks after the last exposure for assessment of pulmonary toxicity. Biomarkers of toxicity assessed in bronchoalveolar fluids showed concentration-dependent changes; biomarkers that showed treatment effects were total cell and neutrophil counts, total protein concentrations and cellular enzymes (lactate dehydrogenase, glutamyl transferase and aspartate transaminase). No statistically significant differences in these biomarkers were detected between rats exposed to air and those exposed to the two low concentrations of lunar dust. Dose-dependent histopathology, including inflammation, septal thickening, fibrosis and granulomas, in the lung was observed at the two higher exposure concentrations. No lesions were detected in rats exposed to ≤6.8 mg/m(3). This 4-week exposure study in rats showed that 6.8 mg/m(3) was the highest no-observable-adverse-effect level (NOAEL). These results will be useful for assessing the health risk to humans of exposure to lunar dust, establishing human exposure limits and guiding the design of dust mitigation systems in lunar landers or habitats.

Comparison of mouse strains and exposure conditions in acute cigarette smoke inhalation studies.

Cigarette smoke exposures in mice have been conducted under various exposure conditions using different strains as animal models of smoke-related diseases. We exposed cigarette smoke to two strains of mice [C57BL/6J (C57) and AKR/J (AKR)] under two different exposure regimens (1 h or 4 h/day) at equivalent daily exposure amount (concentration × time). After 2 weeks exposure, mice were evaluated using exposure markers and biological responses. Smoke exposure suppressed respiratory parameters dependent on exposure concentration. The 1-h regimen groups generally showed a greater degree of respiratory suppression and relatively lower exposure markers of urinary nicotine metabolites than the corresponding 4-h regimen groups. Tidal volume was more suppressed in AKR compared to C57, while respiratory rate was more suppressed in C57. Plasma exposure markers and respiratory parameters suggested that C57 inhaled more volume of smoke than AKR. Changes in bronchoalveolar lavage fluid (BALF) cytology and enzyme parameters were most noticeable in the 1 h AKR groups. In BALF cytokine concentration, TARC concentration in C57 was higher than AKR, while KC and MCP-1 in AKR were higher than C57. Relative lung/body weight ratio in smoke-exposed C57 was generally higher, as well as the incidence and severity of lesions in respiratory organs compared to AKR. In summary, C57 appeared to inhale relatively more smoke and displayed greater inflammatory changes in respiratory tract than AKR. Comparison of exposure regimens suggests that a longer exposure duration at lower WTPM concentration might deliver a larger dose of smoke than a shorter exposure duration at higher WTPM concentration.

Comparison of the physiological and morphological effects of cigarette smoke exposure at comparable weekly doses on Sprague-Dawley rats.

A variety of dose x duration exposure regimens have been used in inhalation toxicity studies using rodents. We evaluated the effects of differences in smoke concentration and daily exposure duration under similar weekly cumulative exposures in rats to determine potential variation in type and severity of adverse effects in 13-week exposure studies. The weekly cumulative dosages were 2100 and 4200 µg wet total particle matter (WTPM)/L, and the daily exposure durations were 1 and 6 h. Weekly exposure duration was 5 or 7 days/week for groups exposed 1 h/day and 7 days/week for groups exposed 6 h/day. Recovery duration was 6 and 13 weeks. Mainstream smoke exposure suppressed body weight (BW) gain in both regimens. Lower dose groups exposed 1 h/day had a consistently greater of BW gain compared with corresponding 6 h/day groups. Respiratory rate, tidal volume, and minute volume (MV) were suppressed in a dose-dependent manner in both regimens. Higher MV in rats exposed for 6 h/day compared with rats exposed 1 h/day suggested that a lower concentration for longer duration resulted in a greater total inhaled mass (TIM) in rats exposed 6 h/day. Groups exposed for 6 h/day had lower blood carboxyhemoglobin and plasma nicotine levels than groups exposed 1 h/day, reflecting the lower carbon monoxide (CO) and WTPM concentrations in the 6 h/day groups. Data from examination of bronchoalveolar lavage fluids (BALF) and respiratory tract tissues indicated comparable effects between both regimens. Exposure-induced histopathological changes regressed similarly for both regimens after the recovery periods.

Proliferative and nonproliferative lesions of the rat and mouse respiratory tract.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the respiratory tract of laboratory rats and mice, with color photomicrographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for respiratory tract lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

1st international ESTP expert workshop: "Larynx squamous metaplasia". A re-consideration of morphology and diagnostic approaches in rodent studies and its relevance for human risk assessment.

Invited international experts participated in a 2-day workshop organized by the European Society of Toxicologic Pathology (ESTP) to evaluate and discuss spontaneous and induced laryngeal lesions in rodents. The main purpose of the workshop was to agree upon the terminology and relevance of a range of laryngeal changes that varied from very subtle epithelial alterations up to severe metaplastic or neoplastic lesions. The workshop experts concluded that minimal, focal epithelial changes of the laryngeal epithelium, predominantly occurring at the base of the epiglottis, should be given the descriptive term of "epithelial alteration" and assessed as "non-adverse". Although observed as induced effects they may also occur in non-treated animals and were not considered to have a potential for a laryngeal dysfunction. Also, cases of minimal to slight laryngeal squamous metaplasia that are not observed diffusely could occur spontaneously or as treatment-induced lesions and should be assessed as "non-adverse". Cases of moderate to severe laryngeal squamous metaplasia observed diffusely in multiple levels should be regarded as "adverse", as there is a potential for dysfunction of the larynx. The occurrence of dysplasia or cellular atypia linked to laryngeal squamous metaplasia should always be reported separately and described in detail. In the evaluation of treatment-related effects of the larynx in studies utilizing aged animals, it has to be considered that moderate or even severe cases of focal laryngeal squamous metaplasia may occasionally be found as age-related, spontaneous lesions. Although inhalation exposure of rodents to non-genotoxic compounds may cause laryngeal squamous metaplasia, none of the workshop experts were aware of any reported cases of tumor induction in the larynx with a non-genotoxic compound. Therefore, for non-genotoxic compounds, the workshop experts did not regard laryngeal squamous metaplasia by itself as a precancerous lesion.

A review of whole animal bioassays of the carcinogenic potential of naphthalene.

This report provides a summary of deliberations conducted under the charge for members of Module A participating in the Naphthalene State-of-the-Science Symposium (NS3), Monterey, CA, October 9-12, 2006. Whole animal bioassays have been performed by the National Toxicology Program in mice and rats to ascertain the carcinogenic potential of naphthalene by inhalation exposure. A statistically significant increased incidence of pulmonary alveolar/bronchiolar adenoma (a benign lesion), was observed among female mice; an observed increase among the males did not reach statistical significance. No nasal tumors were observed in either sex. A tumorigenic response was observed in both sexes of rats, in males an increased incidence of nasal respiratory epithelium adenoma (a benign rather than malignant lesion) and in females, olfactory epithelial neuroblastoma. Interpretations of these studies vary. On the one hand, evidence of extensive non-neoplastic response in both sexes of both species indicates cytotoxicity occurred at all doses, and strongly suggests that cytotoxicity played a significant role in the tumor responses observed in the target tissues. On the other hand, olfactory epithelial neuroblastoma has rarely been observed in NTP bioassays. This review seeks to develop a consensus understanding of the scientific evidence provided by these studies, taking into account that they have been used as the basis for quantitative human cancer risk assessment, and suggests scientific studies that, if performed, could resolve scientific uncertainties.

Pulmonary inflammation in mice exposed to mainstream cigarette smoke.

Male C57Bl/6 (C57) and ICR mice were exposed by nose-only inhalation to mainstream cigarette smoke (MS) from 2R4F reference cigarettes, at concentrations of 75, 250, and 600 microg of total particulate matter (TPM) per liter, for up to 6 mo. Respiratory-tract tissue (nose, larynx, and lung), blood, and bronchoalveolar lavage fluid (BALF) samples were collected and analyzed at several time points. Blood samples were analyzed for biomarkers of exposure (COHb and nicotine). BALF was analyzed for biomarkers of cell injury, inflammation, oxidative stress, enzyme activity, and cytokines. Blood COHb and plasma nicotine concentrations increased in a dose-dependent manner, confirming smoke exposure. Mild emphysema was observed following 28 wk of exposure. Macrophage accumulation and inflammatory infiltrates were observed around the alveolar ducts and adjacent vasculature. There was an approximately 13% increase in mean linear intercept (Lm) only in ICR mice exposed to 600 microg/L TPM. There were no significant changes in biomarkers of oxidative stress secondary to smoke exposures; however, 8-isoprostane significantly increased following the 13-wk post-inhalation period. BALF macrophage and neutrophil counts were rapidly and consistently elevated, while lymphocyte counts gradually increased over time. MS-induced inflammatory responses observed in this study are comparable to changes reported in chronic smokers, supporting the role of chronic inflammation in the pathogenesis of emphysema. However, mild emphysema in minimal numbers of mice suggests that MS exposure concentration and/or duration in the current study were not sufficient to induce a definitive emphysema phenotype.

Upper respiratory tract lesions in inhalation toxicology.

This paper describes some important differences in normal histology of the upper respiratory tract of laboratory animals. It also provides examples of lesions observed or reported in the upper respiratory tract of laboratory animals, predominantly rodents, exposed via inhalation. The anatomy and physiology of upper respiratory tract tissues play a major role in the response to an insult, given that different epithelial types vary in susceptibility to injury and toxicant exposure concentrations throughout the airway vary due to airflow dynamics. Although dogs and nonhuman primates are utilized for inhalation toxicology studies, less information is available regarding sites of upper respiratory injury and types of responses in these species. Awareness of interspecies differences in normal histology and zones of transition from squamous to respiratory to olfactory epithelium in different areas of the upper respiratory tract is critical to detection and description of lesions. Repeated inhalation of chemicals, drugs, or environmental contaminants induces a wide range of responses, depending on the physical properties of the toxicant and concentration and duration of exposure. Accurate and consistent fixation, trimming, and microtomy of tissue sections using anatomic landmarks are critical steps in providing the pathologist the tools needed to compare the morphology of upper respiratory tract tissues from exposed and control animals and detect and interpret subtle differences.

3-week inhalation exposure to cigarette smoke and/or lipopolysaccharide in AKR/J mice.

AKR/J mice were exposed to cigarette smoke (CS) and/or lipopolysaccharide (LPS) via inhalation for 3 wk and pulmonary responses were evaluated. The objective was to explore the feasibility of coexposing LPS with cigarette smoke under a subacute exposure, as a surrogate for viral or bacterial insults, that would mimic the pathogenesis of infection-related chronic obstructive pulmonary disease (COPD) exacerbations. The study was the first step in an effort to develop a rodent COPD model in which morphologic lesions of COPD develop in a shorter period of exposure and more closely simulate human COPD. Mice were exposed 6 h/day, 5 days/wk for 3 wk to one of the following: (1) sham control: filtered air; (2) CS: 250 microg/L wet total particulate matter (WTPM) for 5 h/day followed by 1 h/day air; (3) LPS: 0.5 microg/L LPS (055:B5 Escherichia coli; 3,000,000 EU/mg) for the last 1 h/day 2 day/wk (following 5 h/day of filtered air); and (4) CS/LPS: CS 5 h/day followed by air or LPS (2 days/wk) for 1 h/day. After the last exposure, animals were necropsied and subjected to bronchoalveolar lavage (BAL) or histopathology. The BAL neutrophil counts were highest in the LPS group, while macrophage counts were higher in the CS/LPS group than other exposed groups. The LPS group displayed the greatest increases in BAL cytokines, while KC (keratinocyte-derived chemokine) and TARC (thymus and activation-regulated chemokine) were highest in the CS group. The CS/LPS group had generally lower cytokine levels relative to the LPS or CS groups, except for the levels of RANTES and G-CSF (granulocyte-colony stimulating factor) comparable to the LPS group. At microscopic examination of lung sections, cellular inflammatory infiltrates were most notable in the CS/LPS group, which had a diffuse, predominantly macrophage infiltrate with fewer neutrophils. The LPS group had predominantly neutrophils in the pulmonary infiltrate and the CS group had a predominantly macrophage infiltrate in alveolar ducts and adjacent alveoli. Apoptotic labeling of lung cells was highest with the CS/LPS group. In summary, the CS/LPS group displayed greater cellular infiltration and apoptotic responses in the lung with an indication of immunosuppressive effects (lower BAL cytokines) than the CS or LPS group, suggesting that the CS/LPS model shows promise to be further explored as an animal model for studying pathogenesis of COPD exacerbations. A longer term study with interim assessments is needed to confirm that the subacute responses observed in the CS/LPS group will result in greater severity of COPD-related pulmonary lesions following prolonged exposures.

Effects of flavoring and casing ingredients on the toxicity of mainstream cigarette smoke in rats.

A series of in vitro and in vivo studies evaluated the potential effects of tobacco flavoring and casing ingredients. Study 1 utilized as a reference control cigarette a typical commercial tobacco blend without flavoring ingredients, and a test cigarette containing a mixture of 165 low-use flavoring ingredients. Study 2 utilized the same reference control cigarette as used in study 1 and a test cigarette containing eight high-use ingredients. The in vitro Ames Salmonella typhimurium assay did not show any increase in mutagenicity of smoke condensate from test cigarettes designed for studies 1 and 2 as compared to the reference. Sprague-Dawley rats were exposed by nose-only inhalation for 1 h/day, 5 days/wk for 13 wk to smoke from the test or reference cigarettes already described, or to air only, and necropsied after 13 wk of exposure or following 13 wk of recovery from smoke exposure. Exposure to smoke from reference or test cigarettes in both studies induced increases in blood carboxyhemoglobin ((COHb)) and plasma nicotine, decreases in minute volume, differences in body or organ weights compared to air controls, and a concentration-related hyperplasia, squamous metaplasia, and inflammation in the respiratory tract. All these effects were greatly decreased or absent following the recovery period. Comparison of rats exposed to similar concentrations of test and reference cigarette smoke indicated no difference at any concentration. In summary, the results did not indicate any consistent differences in toxicologic effects between smoke from cigarettes containing the flavoring or casing ingredients and reference cigarettes.

Types and patterns of response in the larynx following inhalation.

The laryngeal mucosa responds to insult similarly to other epithelial tissues but the response depends on location within the larynx since important anatomic differences exist, even within rodent species. Although dogs and nonhuman primates are also utilized for inhalation toxicology studies, little published information is available regarding sites of injury from inhaled toxicants in these species. Accurate and consistent fixation, trimming, and microtomy of laryngeal sections allow the pathologist to compare the morphology of laryngeal mucosa from exposed and control animals and detect and interpret subtle differences resulting from inhalation exposure. There are anatomic landmarks that are keys to providing consistent sections through important areas of the laryngeal mucosa. Repeated inhalation of toxic concentrations of chemicals, drugs, or environmental contaminants induces a wide range of responses, depending on the physical properties and concentration of the toxic substance and duration of exposure. Responses include edema, acute to chronic inflammation, fibrosis, mucosal ulceration, degeneration, and necrosis. Attempts at repair include regeneration, hyperplasia, squamous metaplasia, hyperkeratosis, and neoplasia. Awareness of normal histology and zones of transition from squamous to respiratory epithelium in different areas of the larynx in different species is critical to avoid confusing normal epithelium with metaplasia or hyperplasia. Microscopic examination of laryngeal mucosa from animals exposed via inhalation and necropsied following a recovery period provides the opportunity to determine the degree of regression or progression of exposure-induced laryngeal lesions.

Characterization of mainstream cigarette smoke-induced biomarker responses in ICR and C57Bl/6 mice.

Pulmonary emphysema is a major component of the morbidity and mortality of chronic obstructive pulmonary disease (COPD). Currently there are no predictive biomarkers for COPD. Initial steps toward identifying potentially predictive biomarkers involve utilizing well-characterized mainstream smoke (MS) exposure conditions (dose-response) to identify changes in biomarkers of effect (inflammation, tissue injury, oxidative stress) in emphysema-susceptible and -resistant mouse strains. C57Bl/6 mice have been reported to develop emphysema when exposed chronically to cigarette smoke, while similarly exposed ICR mice do not. Male C57Bl/6 and ICR mice were exposed 2 h/day for 7 consecutive days to MS from a standard reference cigarette (2R4F) at 75, 250, and 600 microg total particulate matter (TPM)/L or filtered air. To confirm exposure, blood samples were collected toward the end of the last exposure and analyzed for carboxyhemoglobin, nicotine, and cotinine. Bronchoalveolar lavage (BAL) fluid samples were collected 2 or 12 h postexposure and analyzed for biomarkers of effect. MS dose differed slightly between strains. More necrosis was observed in nasal epithelium of exposed C57Bl/6 mice. Exposure concentration-dependent increases in apoptosis, chemokines, and neutrophil counts were greater in ICR mice. Similar increases in thymus and activated-regulated chemokine were only observed in C57Bl/6 mice. BAL fluid cells of C57Bl/6 mice appear to undergo necrosis, while the BAL fluid cells of ICR mice appear to undergo apoptosis following MS exposure. Utilizing two strains of mice we identified MS-responsive biomarkers of effect that may be predictive of COPD pathology. Chronic MS exposures are needed to link these biomarkers with emphysema.

Development of alpha2u-globulin nephropathy and adrenal medullary pheochromocytomas in male rats following exposure to Stoddard solvent IIC.

Stoddard solvent IIC is widely used as a solvent in paints and varnishes, and for dry cleaning and other grease removal applications. Because concern exists regarding the long-term effects of occupational exposure in industrial settings, the toxicity and carcinogenicity of Stoddard solvent IIC were evaluated in male and female F344/N rats and B6C3F1 mice. Rats and mice were exposed to 0, 138, 275, 550, 1100, or 2200 mg/m3 Stoddard solvent IIC by whole-body inhalation for 3 mo, and to 0, 138 (male rats), 550, 1100, or 2200 (female rats and male and female mice) mg/m3 for 2 yr. The kidney, liver, and adrenal medulla were targets of Stoddard solvent IIC toxicity in rats. After 3 mo of exposure, male rats developed lesions characteristic of alpha2u-globulin nephropathy. Male and female rats displayed increased liver weights and/or clinical pathology changes suggestive of hepatic injury, although no accompanying histopathologic changes were observed. After 2 yr, increased incidences of adrenal medullary pheochromocytomas provided some evidence of carcinogenicity in male rats. Renal tubule adenomas were slightly increased in male rats after 2 yr, and may have been related to exposure. In mice, there was no chemical-related toxicity after 3 mo, with the exception of increased liver weights in male mice exposed to 2200 mg/m3. After 2 yr, the incidences of hepatocellular adenomas were increased in female mice exposed to 2200 mg/m3; however, these increases were marginal and associated with increases in body weight. There was no evidence of Stoddard solvent IIC carcinogenicity in female rats or male mice. In summary, inhalation exposures of Stoddard solvent IIC resulted in renal toxicity and adrenal medullary pheochromocytomas in male rats. The liver also appeared to be a site of toxicity in male and female rats and mice.

Inhalation toxicology and carcinogenesis studies of propylene glycol mono-t-butyl ether in rats and mice.

Propylene glycol mono-t-butyl ether (PGMBE) is used as a solvent in a variety of commercial applications. Male and female F344/N rats and B6C3F(1) mice were exposed to PGMBE by whole-body inhalation for 2 or 14 weeks (0, 75, 150, 300, 600, or 1200 ppm) or 2 years (0, 75, 300, or 1200 ppm); male NBR rats were exposed for 2 weeks. The kidney and the liver were targets of PGMBE toxicity in rats. Renal lesions suggestive of alpha(2u)-globulin nephropathy were observed in male F344/N, in the 2 and 14-week studies, no kidney lesions were seen in NBR rats. In the 2-year study, male rats displayed exposure-related nonneoplastic lesions in the kidney, and may have shown marginal increases in tubular neoplasms. In the liver, the incidences of hepatocellular adenomas occurred with a positive trend in male rats, and may have been related to PGMBE exposure. In mice of both sexes, the major target of PGMBE toxicity was the liver. In the 2-week study, liver weights and in the 14-week study, liver weights and the incidences of centrilobular hypertrophy were increased. In the 2-year study, the incidences of exposure-related hepatocellular adenoma, adenoma or carcinoma combined, and hepatoblastoma occurred with a positive trend, and were significantly increased in 1200 ppm groups. In summary, exposure to PGMBE resulted in nonneoplastic lesions of the kidney characteristic of alpha(2u)-globulin nephropathy, and may have increased renal tubular neoplasms in male rats. Exposure to PGMBE also produced increases in hepatic tumors in male and female mice.

Alpha 2u-globulin nephropathy and carcinogenicity following exposure to decalin (decahydronaphthalene) in F344/N rats.

Decalin (decahydronaphthalene) is a widely used industrial solvent known to cause male rat-specific alpha2u-globulin nephropathy. In this project, 13-week and two-year inhalation studies of decalin were conducted consecutively in both sexes of F344/N rats. The key objectives were to (1) characterize the 13-week toxicity of decalin in rats, with an emphasis on nephropathy in males; (2) compare the kidney concentrations of decalin, 2-decalone, and alpha2u-globulin in males over 2 to 13 weeks of decalin exposure; and (3) correlate male rat nephropathy observed in the 13-week study with renal carcinogenicity in the two-year study. F344 rats (M/F) were exposed via whole-body inhalation to 0, 25, 50, 100, 200, or 400 ppm decalin for 13 weeks. Urine was collected at weeks 2 and 6 for creatinine and decalol analyses and at week 12 for clinical urinalysis. Right kidneys were collected from male rats at weeks 2 and 6 and from both sexes at week 13, homogenates were prepared using the whole kidney, and these homogenates were analyzed for alpha2u-globulin, decalin, and 2-decalone. Left kidneys were evaluated for histopathology and cell proliferation utilizing a proliferating cell nuclear antigen technique and counting proximal renal tubular epithelial cells to determine cell labeling indices. Necropsies and histopathologic evaluations were performed at week 13. Decalin exposure caused increases in kidney weight, urinalysis parameters (protein, AST, LDH), kidney alpha2u-globulin concentration, and proximal convoluted renal tubular cell proliferation in males. These changes were accompanied by microscopic lesions (accumulation of hyaline droplets in cortical tubules, regeneration of proximal tubular epithelium, and granular casts in medullary tubules) clearly linked to alpha2u-globulin nephropathy. Both decalin and 2-decalone were related to increased alpha2u-globulin in male kidneys. Kidney concentrations of decalin, 2-decalone, and alpha2u-globulin in exposed females were negligible, while females excreted greater amounts of decalol metabolites in urine than males at weeks 2 and 6. There were no exposure-related microscopic lesions in females. For chronic exposure, F344 rats were exposed via whole-body inhalation to 0, 25, 50 (males only), 100, or 400 ppm decalin for two years. Chronic exposure induced a spectrum of nonneoplastic and neoplastic lesions in the renal cortex of males, ranging from regenerative lesions of chronic nephropathy to tubular carcinomas. Incidences of renal tubular adenoma, tubular carcinoma, combined tubular adenomas and carcinomas, cortical tubular hyperplasia, hyaline droplet accumulation, hyperplasia of pelvic epithelium, and mineralization in renal papilla were increased in exposed males compared to controls. There was a clear increase in the mean severity of chronic nephropathy in decalin-exposed males. It was concluded that the carcinogenic effect on the renal cortical epithelium of male rats exposed to decalin was related to increased turnover of this epithelium, resulting from the cytotoxic effects of alpha2u-globulin accumulation in the renal cortical tubular cell cytoplasm.