Subchronic pulmonary pathology, iron overload, and transcriptional activity after Libby amphibole exposure in rat models of cardiovascular disease.

BACKGROUND: Surface-available iron (Fe) is proposed to contribute to asbestos-induced toxicity through the production of reactive oxygen species. OBJECTIVE: Our goal was to evaluate the hypothesis that rat models of cardiovascular disease with coexistent Fe overload would be increasingly sensitive to Libby amphibole (LA)-induced subchronic lung injury. METHODS: Male healthy Wistar Kyoto (WKY), spontaneously hypertensive (SH), and SH heart failure (SHHF) rats were intratracheally instilled with 0.0, 0.25, or 1.0 mg LA (with saline as the vehicle). We examined bronchoalveolar lavage fluid (BALF) and histological lung sections after 1 week, 1 month, or 3 months for pulmonary biomarkers and pathology. SHHF rats were also assessed at 6 months for pathological changes. RESULTS: All animals developed concentration- and time-dependent interstitial fibrosis. Time-dependent Fe accumulation occurred in LA-laden macrophages in all strains but was exacerbated in SHHF rats. LA-exposed SHHF rats developed atypical hyperplastic lesions of bronchiolar epithelial cell origin at 3 and 6 months. Strain-related baseline differences existed in gene expression at 3 months, with persistent LA effects in WKY but not SH or SHHF rats. LA exposure altered genes for a number of pathways, including inflammation, immune regulation, and cell-cycle control. Cell-cycle control genes were inhibited after LA exposure in SH and SHHF but not WKY rats, whereas tumor suppressor genes were induced only in WKY rats. The inflammatory gene expression also was apparent only in WKY rats. CONCLUSION: These data show that in Fe-overload conditions, progressive Fe accumulation occurs in fiber-laden macrophages within LA-induced lesions. Fe overload does not appear to contribute to chronic inflammation, and its role in hyperplastic lesion development requires further examination.

Pulmonary inflammatory and fibrotic responses in Fischer 344 rats after intratracheal instillation exposure to Libby amphibole.

Increased incidences of asbestosis have been reported in workers from Libby, MT, associated with exposures to amphibole-contaminated vermiculite. In this study pulmonary and histopathological changes were investigated following Libby amphibole (LA) exposure in a rat model. Rat respirable fractions of LA and amosite (aerodynamic diameter <2.5 µm) were prepared by water elutriation. Male F344 rats were exposed to single doses of either saline (SAL), amosite (0.65 mg/rat), or LA (0.65 or 6.5 mg/rat) by intratracheal instillation. At times from 1 d to 3 mo after exposure, bronchoalveolar lavage (BAL) was performed and right and left lungs were removed for reverse-transcription polymerase chain reaction (RT-PCR) and histopathological analysis, respectively. Data indicated that 0.65 mg amosite resulted in a higher degree of pulmonary injury, inflammation, and fibrotic events than LA at the same mass dose. Exposure to either amosite or high dose LA resulted in higher levels of cellular permeability and injury, inflammatory enzymes, and iron binding proteins in both BAL fluid and lung tissue at most time points when compared to SAL controls. However, mRNA expression for some growth factors (e.g., platelet-derived growth factor [PDGF]-A and transforming growth factor [TGF]-1ß), which contribute to fibrosis, were downregulated at several time points. Furthermore, histopathological examination showed notable thickening of interstitial areas surrounding the alveolar ducts and terminal bronchioles. On a mass dose basis, amosite produced a greater acute and persistent lung injury for at least 3 mo after exposure. However, further testing and analysis of LA are needed with regard to the dose metric to fully evaluate its potential fibrogenicity and carcinogenicity.

Pulmonary oxidative stress, inflammation, and dysregulated iron homeostasis in rat models of cardiovascular disease.

Underlying cardiovascular disease (CVD) is a risk factor for the exacerbation of air pollution health effects. Pulmonary oxidative stress, inflammation, and altered iron (Fe) homeostasis secondary to CVD may influence mammalian susceptibility to air pollutants. Rodent models of CVD are increasingly used to examine mechanisms of variation in susceptibility. Baseline cardiac and pulmonary disease was characterized in healthy normotensive Wistar Kyoto (WKY) rats, cardiovascular compromised spontaneously hypertensive rats (SHR), and spontaneously hypertensive heart failure (SHHF) rats. Blood pressure, heart rate, and breathing frequencies were measured in rats 11 to 12 wk of age, followed by necropsy at 14 to 15 wk of age. Blood pressure and heart rate were increased in SHR and SHHF relative to WKY rats (SHR > SHHF > WKY). Increased breathing frequency in SHHF and SHR (SHR > SHHF > WKY) resulted in greater minute volume relative to WKY. Bronchoalveolar lavage fluid (BALF) protein and neutrophils were higher in SHHF and SHR relative to WKY (SHHF >> SHR > WKY). Lung ascorbate and glutathione levels were low in SHHF rats. BALF Fe-binding capacity was decreased in SHHF relative to WKY rats and was associated with increased transferrin (Trf) and ferritin. However, lung ferritin was lower and Trf was higher in SHHF relative to WKY or SHR rats. mRNA for markers of inflammation and oxidative stress (macrophage inflammatory protein [MIP]-2, interleukin [IL]-1alpha, and heme oxygenase [HO]-1) were greater in SHHF and SHR relative to WKY rats. Trf mRNA rose in SHR but not SHHF relative to WKY rats, whereas transferrin receptors 1 and 2 mRNA was lower in SHHF rats. Four of 12 WKY rats exhibited cardiac hypertrophy despite normal blood pressure, while demonstrating some of the pulmonary complications noted earlier. This study demonstrates that SHHF rats display greater underlying pulmonary complications such as oxidative stress, inflammation, and impaired Fe homeostasis than WKY or SHR rats, which may play a role in SHHF rats' increased susceptibility to air pollution.

Pulmonary and systemic effects of zinc-containing emission particles in three rat strains: multiple exposure scenarios.

As a common component of ambient particulate matter (PM), zinc has been proposed to play a role in PM-induced adverse health effects. Although occupational exposures to high levels of zinc-fume have been associated with metal-fume fever accompanied by pulmonary inflammation and injury, the effects of PM-associated zinc are unclear. We hypothesized that an oil combustion emission PM (EPM) containing bioavailable zinc would induce pulmonary injury and systemic hematological changes attributable to the leachable zinc following acute as well as longer-term exposures in a rat strain-specific manner. In order to initially characterize the pulmonary response to EPM, male Sprague-Dawley (SD) rats were intratracheally (IT) instilled with 0.0, 0.8, 3.3, or 8.3 mg/kg EPM in saline. To further determine if the pulmonary injury was associated with the EPM leachable zinc, subsequent studies included IT instillation of SD rats with either saline, whole EPM suspension, the saline leachable fraction of EPM, the particulate fraction of EPM (all at 8.3 mg/kg, soluble Zn = 14.5 microg/mg EPM), or ZnSO(4) (0.0, 33.0, or 66.0 microg/kg Zn). Finally, to ascertain the cumulative impact of inhaled EPM in the causation of acute pulmonary and systemic effects as well as long-term fibrotic responses, we exposed three rat strains of differential susceptibility to PM. Male SD, normotensive Wistar-Kyoto (WKY), and spontaneously hypertensive (SH) rats (90 days old) were exposed nose-only to either filtered air or EPM: 2, 5, or 10 mg/m(3) (6 h/day x 4 days/week x 1 week); or 10 mg/m(3) (6 h/day x 1 day/week for 1, 4, or 16 weeks) and assessed at 2 days postexposure. IT exposures to whole EPM suspensions were associated with a dose-dependent increase in protein/albumin permeability and neutrophilic inflammation. Pulmonary protein/albumin leakage and neutrophilic inflammation caused by the leachable fraction of EPM and ZnSO(4) were comparable to the effect of whole suspension. However, protein/albumin leakage was not associated with the particulate fraction, although significant neutrophilic inflammation did occur following instillation. With EPM nose-only inhalation, acute exposures (10 mg/m(3) only) for 4 days resulted in small increases in bronchoalveolar lavage fluid (BALF) protein and n-acetyl glucosaminidase activities (approximately 50% above control). Surprisingly, unlike IT exposures, no neutrophilic influx was detectable in BALF from any of the inhalation groups. The only major effect of acute and long-term EPM inhalation was a dose- and time-dependent increase in alveolar macrophages (AM) regardless of the rat strain. Histological evidence also showed dose- and time-dependent accumulations of particle-loaded AM. Particles were also evident in interstitial spaces, and in the lung-associated lymph nodes following the inhalation exposures (SH > WKY = SD). There were strain-related differences in peripheral white blood cell counts and plasma fibrinogen with no major EPM inhalation effect. The present study demonstrated the critical differences in pulmonary responsiveness to EPM between IT and inhalation exposures, probably attributable to the dose of bioavailable zinc. EPM IT exposures, but not acute and long-term inhalation of up to 10 mg/m(3), caused neutrophilic inflammation. Inhalation exposures may result in particle accumulation and macrophage recruitment with potential strain differences in EPM clearance.

Cardiac and thermoregulatory effects of instilled particulate matter-associated transition metals in healthy and cardiopulmonary-compromised rats.

Particulate matter air pollution has been associated with cardiopulmonary morbidity and mortality in many recent epidemiological studies. Previous toxicological research has demonstrated profound cardiac and thermoregulatory changes in rats following exposure to residual oil fly ash (ROFA), a combustion-derived particulate. The response to ROFA appeared biphasic, consisting of both immediate (0-6 h) and delayed (24-96 h) bradycardia and hypothermia. Other studies have demonstrated that much of the pulmonary toxicity of ROFA was caused by its constitutive transition metals, namely, Fe, Ni, and V. This study examined the contributions of these metals to the observed cardiac and thermoregulatory changes caused by ROFA in conscious, unrestrained rats. Prior to exposure, each animal was surgically implanted with a radiotelemetry device capable of continuously monitoring heart rate, electrocardiographic, and core temperature data. Individual metals were intratracheally instilled in healthy rats (n = 4 per metal species) and in rats with monocrotaline (MCT; 60 mg/kg)-induced pulmonary hypertension (n = 10 per metal species); combinations of metals were instilled in MCT-treated rats only (n = 6 per combination of metal species). Metals were administered in doses equivalent to those found in the highest dose of ROFA used in previous studies, that is, 105 microg Fe(2)(SO(4))(3), 263 microg NiSO(4), and 245 microg VSO(4). Healthy and MCT-treated rats demonstrated similar responses to metals. Fe caused little response, whereas V caused marked bradycardia, arrhythmogenesis, and hypothermia immediately following instillation and lasting approximately 6 h. Ni caused no immediate response, but induced a delayed bradycardia, arrhythmogenesis, and hypothermia that began approximately 24 h after instillation and lasted for several days. When instilled in combination, Ni appeared to exacerbate the immediate effects of V, whereas Fe attenuated them. These data suggest that the biphasic response to instilled ROFA may result from a summation of the temporally different effects of V and Ni.